Mesenchymal-like stem cells derived from human embryonic stem cells, methods and uses thereof

ABSTRACT

The disclosure provided herein relates generally to mesenchymal-like stem cells “hES-T-MiSC” or “T-MSC” and the method of producing the stem cells. The method comprises culturing embryonic stem cells under conditions that the embryonic stem cells develop through an intermediate differentiation of trophoblasts, and culturing the differentiated trophoblast in hES-T-MSC or T-MSC, T-MSC derived cells and cell lineages “T-MSC-DL” are also described. Disclosed also herein are solutions and pharmaceutical compositions comprising the T-MSC and/or T-MSC-DL, methods of making the T-MSC and T-MSC-DL, and methods of using the T-MSC and T-MSC-DL for treatment and prevention of diseases, specifically. T-MSC and T-MSC-DL are used as immunosuppressive agents to treat multiple sclerosis and autoimmune diseases.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationSer. No. 61/670,192, filed Jul. 11, 2012 and U.S. ProvisionalApplication Ser. No. 61/684,509, filed Aug. 17, 2012, which are herebyincorporated by reference in their entireties

INTRODUCTION

The disclosure provided herein relates generally to mesenchymal-likestem cells “hES-T-MSC” or “T-MSC” and the method of producing the stemcells. The method comprises culturing embryonic stem cells underconditions that the embryonic stem cells develop through an intermediatedifferentiation of trophoblasts, and differentiating trophoblasts intohES-T-MSC or T-MSC. Disclosed herein are the T-MSC, solutions andpharmaceutical compositions comprising the T-MSC, methods of making theT-MSC, methods of using the T-MSC for treatment and prevention ofdiseases, specifically, T-MSC are used as an immunosuppressive agent totreat multiple sclerosis and other autoimmune diseases, for tissueregeneration/repair uses, and methods of using the T-MSC for thedelivery of agents across the blood brain barrier and the blood spinalcord barrier. Also disclosed herein are methods of using T-MSCs tomodulate the immune system, inhibit immune response to an individual'sself-antigen and repair damaged central nervous systems. Compositionscomprising T-MSCs for use in immunomodulation are disclosed herein, asare methods of providing modified T-MSC with improved immunosuppressivefunction through modified gene expression.

BACKGROUND

Human mesenchymal stem/stromal cells (MSCs) have been widely used forimmune system regulation and tissue repair. Human embryonic stem cells(hESCs) can be used as a reliable source for generating high-qualityhuman MSCs. There are many methods to differentiate hESCs into MSCs.However, current methods are not able to conduct such differentiation inan efficient manner to produce a high yield of high purity MSCs.

Mesenchymal stem cells (MSCs) derived from adult mouse or human tissuessuch as bone marrow, umbilical cord and fat tissue are multipotent i.e.,capable of generating a variety of mature cell lineages includingadipocytes, chondrocytes, osteoblast cells, neural lineage cells,myoblast, stromal cells and fibroblast, etc. These technologies havebeen well characterized and patented. For example, see Caplan et al.,U.S. Pat. No. 5,466,359 (human mesenchymal stem cells).

However, the currently available adult tissue-derived MSCs have severalpitfalls. First, the limited sources and varying quality of the donortissues such as the bone marrow restrict the study and application ofthe MSCs and prevent the standardization of the MSCs as a medicalproduct for large-scale clinical use. Second, the MSCs obtained from theadult tissues are highly mixed populations of cells, in which only asmall portion of the cells have strong immunosuppressive effect, Toobtain enough cell numbers for clinical use, in vitro expansion isnecessary, which can decrease the immunosuppressive and horningabilities of MSCs (Javazon et al., 2004). Third, there are safety issuesregarding to the use of adult-derived MSCs including malignanttransformation (Wong, 2011) and potential transmission of infectiouspathogens from donors.

To overcome these pitfalls, scientists have attempted to derive MSCsfrom hESCs via various methods. These methods involve either co-culturewith the mouse OP9 cell line or handpicking plus the use of multiplecytokines and chemicals (Barberi et al., 2005; Chen et al., 2012; Liu etal., 2012; Sanchez et al., 2011). Recently, a TGFβ signaling inhibitorSB431542 has been used to differentiate hESCs into MSCs, whichsimplifies the procedures and improves the efficiency (Chen et al.,2012), but the yield and purity are quite low (see the below-describedcomparison tests.). In 2010, the inventors and Advanced Cell Technologydeveloped another method to derive MSC from hemangioblast, whichinvolved the use of many expensive cytokines and methylcellulose medium,but the derivation efficiency is also low using this method,

Currently known methods for differentiation of hESCs into MSCs are eachcharacterized as having one or more serious shortcomings and weaknessesDifferentiation of MSCs from hESCs co-cultured with the OP9 stromalcells has the disadvantages of being time consuming, producing cells oflow yield, low purity, and using animal feeder cells and undefinedculture conditions (Barberi et al., 2005). Differentiation fromoutgrowing cells around replated embryoid bodies formed by hESCs has thedisadvantages of being time consuming, producing cells in low yield,using undefined culture condition, and being an expensive method(Olivier et al., 2006). Differentiation from hESCs cultured oncollagen-coated plates has the disadvantages of very low yield,undefined culture conditions, and being time consuming (Liu et al.,2012). Differentiation with hESCs treated with inhibitors of TGFβsignaling has the disadvantages including low purity of cells (per ourtests), low cell yield, time consuming method, and low immunosuppressiveeffect of the cells that are produced (Chen et al., 2012; Sanchez etal., 2011). Thus, there is a need for an unlimited, safe, highly stable,efficient and consistent source of MSCs to use as a treatment andprophylactic for various diseases.

Multiple sclerosis (MS) is a chronic autoimmune disease caused byinfiltration of peripheral immune cells into the central nervous system(CNS) through damaged blood-brain barrier (BBB) or blood-spinal cordbarrier (BSCS), which causes inflammation of the myelin sheaths aroundneuronal axons, and causes demyelination and scarring of the axons(McFarland and Martin (2007)). According to the National MultipleSclerosis Society of United States, there are more than 70 FDA-approvedmedications for the treatment of MS, including Avonex (IFNβ-1a),Betaseron (IFNβ-1b), Gilenya (a sphingosine 1-phosphate receptormodulator). Glatiramer acetate (or Copolymer 1), and Tysabri (humanizedanti-α-integrin antibody). However, these offer only palliative reliefand are associated with serious adverse effects including increasedinfection, heart attack, stroke, progressive multifocalleukoencephalopathy, arrhythmia, pain, depression, fatigue, maculaedema, and erectile dysfunction (Johnston and So (2012); Weber et al.(2012)).

Transplantation of mesenchymal stromal/stem cells (MSCs) has emerged asa potentially attractive therapy due to their immunomodulatory andneuroregenerative effects (Auletta et al., (2012); Pittenger et al.(1999)) and potential ability to repair the blood-brain barrier (Chao etal. (2009); Menge et al. (2012)). MSCs are multipotent meaning they cangenerate a variety of cell lineages including adipocyte, chondrocyte,osteoblast cells and neurons. They can be derived from fetal, neonatal,and adult tissues such as the amniotic membrane, umbilical cord, bonemarrow, and adipose. MSCs have several unique advantages over currentpharmacotherapies, as these cells can serve as carriers of multiple andpotentially synergistic therapeutic factors, and can migrate to injuredtissues to exert local effects through secretion of mediators andcell-cell contact (Uccelli and Prockop (2010a)). Importantly, MSCs havebeen found efficacious in the treatment of mice with experimentalautoimmune encephalomyelitis (EAE), a well-recognized animal model of MS(Gordon et al., 2008a; Gordon et al. (2010); Morando et al. (2012);Peron et al. (2012); Zappia et al. (2005); Zhang et al. (2005)), as wellas MS patients in clinical trials (Connick et al. (2012); Karussis etal. (2010); Mohyeddin Bonab et al. (2007); Yamout et al. (2010)).Xenogeneity does not appear problematic as both mouse and human bonemarrow-derived MSC (BM-MSC) can attenuate disease progression of EAEmice (Gordon et al. (2008a); Gordon et al. (2010); Morando et al.(2012); Peron et al. (2012); Zappia et al. (2005); Zhang et al. (2005)).However, varying effects were reported on EAE mice treated with BM-MSCin different reports (Gordon et al. (2008a); Payne et al. (2012); Zappiaet al. (2005); Zhang et al. (2005)). The efficacy of BM-MSC on treatmentof the disease is questionable.

There is a strong need for an unlimited, safe, highly stable, efficientand consistent source of MSC to use as a treatment and prophylactic forthese diseases as well as others. Disclosed herein are hES-T-MSCsderived from hESCs through a highly efficient differentiation methodthat meets these needs. Also disclosed herein are a microarray analysisand other analysis, where several key factors are identified that aredifferentially expressed in hES-T-MSC compared to BM-MSC and otherhES-MSC differentiated through other methods.

SUMMARY

Disclosed herein is a method to derive mesenchymal-like stem cells fromhESCs through an intermediate step of trophoblast induction. The MSCsderived via this method are called “hES-T-MSC” or “T-MSC”. The T-MSC maybe differentiated into cells or cell lineages including, but not limitedto, adipocytes, myoblast cells, neuron cells, osteoblast cells,fibroblast, chondrocytes, stromal cells The T-MSC derived cells or celllineages or called “T-MSC derived lineages” or “T-MSC-DL”.

Disclosed herein are compositions, including compositions comprisingT-MSC and/or T-MSC-DL, having immunosuppressive properties. Describedherein are populations of T-MSC and/or T-MSC-DL selected on the basis oftheir ability to modulate an immune response, and compositions havingimmunomodulatory properties. As disclosed herein, T-MSC and/or T-MSC-DLhave higher immunosuppressive activity compared to bone marrow-derivedMSCs.

Disclosed herein is a method to efficiently produce T-MSC in high purityand high yield. The method has the features of relatively few steps andfewer required differentiation factors than previously reported.

Disclosed herein are methods of using human embryonic stem cells (hESCs)to derive mesenchymal-like stem cells through an intermediatedifferentiation of trophoblasts. The MSCs derived from trophoblasts arecalled hES-T-MSC or T-MSC. The T-MSC can be used to modulate the immunesystem. For example, they are effective in treating multiple sclerosisby preventing immune cell-caused damage in the central nervous systems.

Disclosed herein are human embryonic-derived mesenchymal stem cellsproduced by the methods disclosed herein.

Disclosed herein are methods to induce differentiation of T-MSC intoT-MSC-DL.

Also disclosed herein is the application of the T-MSC and/or T-MSC-DL totreat multiple sclerosis and other autoimmune diseases in mammals andespecially in human subjects.

It is a further object of the disclosed invention to provide a cellproduct T-MSC for use in immunomodulation, for example, for preventionor inhibition of immunorejection during tissues or organtransplantation. In another specific embodiment of the method ofreducing or suppressing an immune response, the immune response isgraft-versus-host disease. In another specific embodiment, the immuneresponse is an autoimmune disease, e.g., diabetes, lupus erythematosus,or rheumatoid arthritis.

It is a further object of the disclosed invention to provide a cellproduct T-MSC-DL for use in treatment of neural diseases.

The method can employ as many stem cells provided herein as are requiredto effect a detectable suppression of an immune response. For example,the plurality of stem cells provided herein used to contact theplurality of immune cells can comprise 1×10⁵ T-MSC, 1×10⁶ T-MSC, 1×10⁷T-MSC, 1×10⁶ T-MSC or more.

In one embodiment, the method described herein is a novel process forderiving (also referred to herein as producing) MSCs from hESCs. Themethod comprising the steps of:

-   -   a. Culturing a cell culture comprising human embryonic stem        cells in serum-free medium in the present of at least one growth        factor in an amount sufficient to induce the differentiation of        the embryonic stem cells to differentiate into trophoblasts; in        an embodiment, the time period of the differentiation into        trophoblasts is about 2-5 days; in an embodiment, the medium        comprises BMP4, with or without the presence of a TGFβ inhibitor        (i.e. SB431542, A83-01 or ALK5 inhibitor, etc.) to increase the        differentiation efficiency:    -   b. Adding at least one growth factor to the culture comprising        the trophoblasts and continuing to culture in serum-free medium,        wherein the growth factor is in an amount sufficient to expand        the trophoblasts, in an embodiment, the medium comprises BMP4        (this step is optional);    -   c. Isolating the trophoblasts and re-plating the trophoblasts        onto gelatin, laminin, fibronectin, vitronectin, collagen or        Matrigel-coated plates and cultured in a serum-containing or        serum-free media in an amount sufficient to differentiate the        trophoblasts into T-MSC through pre-T-MSC, in an embodiment, the        isolated trophoblasts are cultured for 4-10 days to produce the        T-MSC, wherein at least about 90%, 95%, 96%, 97%, 98%, 99% of        the resulting T-MSC express cell surface markers for adult MSCs,        in an embodiment, the medium comprises LIF, bFGF, or PDGF to        increase expansion efficiency.

In a specific embodiment, the trophoblasts derived from hESC expressTrop-2, but not CD73.

In a specific embodiment, the pre-T-MSC express Trop-2 and/or CD73.

In a specific embodiment, the T-MSC express CD73⁺CD105³⁰ CD90⁺. It is anobject of the disclosed method to differentiate hESCs into MSCs of highpurity. In a preferred embodiment, CD73⁺CD105³⁰ CD90⁺ T-MSC are producedwith greater than 90%, 95%, 96%, 97%, 98%, 99% purity.

A large number of T-MSG with high purity is demonstrated by theobservation that high percentages of the MSCs express cell-surfacemarkers for adult MSCs. The MSCs have higher immunosuppressive effectboth in vitro and in vivo than MSCs obtained via other methods. The MSCsderived via this currently disclosed method are namedhES-trophoblast-derived MSCs and are more briefly referred to herein asT-MSC.

In certain embodiments, the serum-containing medium contains fetal calfserum or human AB serum, L-glutamine and the serum-free medium containsknockout serum replacement (KOSR) or bovine serum albumin (BSA).

In certain embodiments, there is an additional step of irradiating theresulting T-MSC with gamma radiation ranging from 1 gy to 200 gy.

In a further embodiment of the current invention, the method forgenerating and expanding T-MSC results in at least 10,000 T-MSC, atleast 50,000 T-MSC, at least 100,000 T-MSC, at least 500,000 T-MSC, atleast 1×10⁶ T-MSC, at least 5×10⁶ T-MSC, at least 1×10⁷ T-MSC, at least5×10⁷ T-MSC, at least 1×10⁹ T-MSC, at least 5×10⁶ T-MSC, at least 1×10⁹T-MSC, at least 5×10⁹ T-MSC, or at least 1×10¹⁰ T-MSC. These methodsresult in cell solutions that may comprise between 10,000 and 10 billionT-MSC, in certain embodiments, at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% of the resulting human embryonic-mesenchymalstem cells express one or more hES-MSC differential markers. In certainembodiments, the marker is CD73, CD90 and CD105.

In one embodiment, the T-MSCs remarkably attenuate the disease score ofthe EAE mice, accompanied by decreased demyelination, T cellinfiltration, and microglial responses. In addition, the T-MSCs navemuch stronger immunosuppressive activity in vivo and in vitro whencompared to bone marrow derived MSCs (BM-MSC). Also provided herein arekey proteins/molecules that are differentially expressed between T-MSCand BM-MSCs. Provided herein are methods of identifying T-MSCs withimproved immunosuppressive activity by measuring the expression level ofthe protein/molecular markers. Also disclosed are methods of geneticmodification to improve immunosuppressive activity of T-MSCs.

A further embodiment of the present invention is a solution comprisingT-MSC comprising at least 10,000 T-MSC, at least 50,000 T-MSC, at least100,000 T-MSC, at least 500,000 T-MSC, at least 1×10⁶ T-MSC, at least5×10⁶ T-MSC, at least 1×10⁷ T-MSC, at least 5×10⁷ T-MSC, at least 1×10⁸T-MSC, at least 5×10⁸ T-MSC, at least 1×10⁹ T-MSC, at least 5×10⁹ T-MSC,or at least 1×10¹⁰ T-MSC.

In certain embodiments, the culture volume is from 2 ml for at least10,000 cells, 10 ml for at least 100,000 cells, 100 ml for at least1,000,000 cells, 1000 ml for at least 10,000,000 cells, and up to 4000ml of media for 5×10⁸ cells.

These solutions can be injected into a subject. These solutions can befrozen. These solutions can be used for the manufacture of a medicamentfor a disease that can be treated by the administration of T-MSC.

This invention also provides a method for producing a solution of T-MSCsuitable for injection into a patient comprising the steps of isolatingthe solution of cells described in the preceding paragraph and placingthe cells into solution suitable for injection into a patient. Thisinvention also provides a method of producing a solution of T-MSCsuitable for freezing comprising the steps of isolating the cellsdescribed in the preceding paragraph and placing into a solutionsuitable for freezing.

Yet another embodiment of the present invention is a T-MSC expressingone or more of cell marker proteins including CD73, CD90, CD105, CD13,CD29, CD54, CD44, CD146, CD166 or a combination thereof. In a furtherembodiment, the human embryonic-mesenchymal stem cell does not expressor expresses low levels of one or more cell marker proteins includingCD34, CD31, CD45 or a combination thereof. In a further embodiment, thehuman embryonic-mesenchymal stem cell does not express or expresses lowlevels of one or more pro-inflammatory proteins including MMP2, RAGE,IFNγR1, IFNγR2, IL-12, TNFα, IL-6, VCAM1 or a combination thereof. Incertain embodiments, the human embryonic-mesenchymal stem cell expressedat least half of the level of the above markers as compared to bonemarrow derived MSC.

A further embodiment of the present invention is a cell culturecomprising T-MSC expressing one or more of cell marker proteins:including CD73, CD90, CD105, CD13, CD29, CD54, CD144, CD146 and CD44. Ina further embodiment, the T-MSC in the cell culture do not express orexpress low levels of one or more cell marker proteins including CD34,CD31 and CD45. In a further embodiment, the T-MSC in the cell culture donot express or express low levels of one or more pro-inflammatoryproteins including MMP2, RAGE, IFNγR1, IFNγR2, TNFα, IL-6, and VCAM1.

In certain embodiments, the cell culture comprises at least 1×10⁶ T-MSC,at least 1×10⁷ T-MSC at least 1×10⁸ T-MSC, at least 1×10⁹ T-MSC, or atleast 1×10¹⁰ T-MSC.

In further embodiments, at least about 90% of the T-MSC in the cellculture express the CD73 protein, at least more than 90% of the T-MSCexpress the CD73 protein, at least about 95% T-MSC express the CD73protein, or more than 95% T-MSC express the CD73 protein. In furtherembodiments, at least about 96% of the T-MSC in the cell culture expressthe CD73 protein, at least more than 97% of the T-MSC express the CD73protein, at least about 98% T-MSC express the CD73 protein, or more than99% T-MSC express the CD73 protein.

In further embodiments, at least about 75%, 80%, 85%, 90%, 95%, 99% ofthe T-MSC in the cell culture express at least one cell marker proteinselected from the group consisting of CD90, CD105, CD44, and CD29.

In further embodiments, at least about 80%, 85%, 90%, 95%, 99% of theT-MSC in the cell culture do not express or express low levels of atleast one cell marker including CD34, CD31, and CD45.

In further embodiments, at least about 75%, 80%, 85%, 90%, 95%, 99% ofthe T-MSC in the cell culture do not express or express low levels of atleast one pro-inflammatory protein including MMP2, RAGE, IFNγR1, IFNγR2,IL-12, TNFα, IL-6, and VCAM1. In certain embodiments, the T-MSC expresshigh levels of CD24, TGFβ2 or both.

In certain embodiments of the T-MSC or cell cultures described herein,the cells are irradiated using gamma radiation.

Further embodiments of the present invention are pharmaceuticalpreparations comprising any one of the T-MSC or cell cultures describedherein and pharmaceutically acceptable carriers.

Yet further embodiments of the present invention are cryopreservedpreparations of any of the T-MSC or cell cultures described herein.

Provided herein are methods of treating or preventing a T cell relatedautoimmune disease in a subject in need thereof, comprising the steps ofadministering a therapeutically effective amount of solution, cellculture or pharmaceutical preparation comprising T-MSC as described inthe preceding paragraphs, to the subject in need thereof. The T cellrelated autoimmune diseases include but are not limited to Crohn'sdisease, inflammatory bowel disease, graft versus host disease, systemiclupus erythematosus, and rheumatoid arthritis, T cell mediated delayedtype hypersensitivity (Type IV hypersensitivity) i.e., Type 1 diabetesmellitus, MS, RA, Hashimoto's thyroiditis, Crohn's, contact dermatitis,Scleroderma, etc.

In certain embodiments, the subject is preferably a mammal or avian, andmost preferably human. In certain embodiments, the solution, cellculture or pharmaceutical preparation comprises irradiated ornon-irradiated T-MSC.

In certain embodiments, the method for treating or preventing diseaseincludes combination therapy with one or more therapeutic agents for thetreatment or prevention of disease.

In other certain embodiments, the present invention provides methods fortreating or preventing multiple sclerosis disease in a subject in needthereof, comprising the steps of administering a therapeuticallyeffective amount of solution, cell culture or pharmaceutical preparationcomprising T-MSC as described in the preceding paragraphs, to thesubject in need thereof. The multiple sclerosis can berelapsing/remitting multiple sclerosis, progressive/relapsing multiplesclerosis, primary multiple sclerosis, or secondary multiple sclerosis.The subject is preferably a mammal, and most preferably human, Thesolution, cell culture or pharmaceutical preparation can compriseirradiated or non-irradiated T-MSC.

The method can further comprise the administration of additionaltherapeutic agents to the subject, including but not limited to,fingolimod, adrenocorticotropic hormone (ACTH), methylprednisolone,dexamethasone, IFNβ-1a, IFN-1b, gliatriamer acetate, cyclophosphamide,methotrexate, azathioprine, cladribine, cyclosporine, mitoxantrone, andsulfasalazine. In yet another embodiment, one or more of thesetherapeutic agents can be attached to the T-MSCs in order to cross theblood-brain and/or blood-spinal cord barrier, for delivery of thetherapeutic agent to the central nervous system.

Provided herein is a method of delivering an agent through theblood-brain barrier and/or the blood-spinal cord barrier, the methodcomprising the steps of attaching or conjugating the agent to a T-MSC toform a complex; and administering the human embryonic-mesenchymal stemcell-agent complex to a subject in need thereof, wherein the T-MSC iscapable of crossing the blood-brain barrier and/or the blood-spinal cordbarrier and the agent is for the treatment, prevention or diagnosis of adisease or injury in the subject in need thereof. T-MSC may be in theform of a single cell, a cell culture, a solution or a pharmaceuticalpreparation. Agents would include, but are not limited to, drugs,proteins, DNA, RNA, and small molecules.

A further embodiment is a delivery system comprising a T-MSC and aconjugated or attached agent, for crossing the blood-brain barrierand/or the blood-spinal cord barrier,

The method described herein has a number of advantages. It is an objectof the disclosed method to differentiate hESCs via an intermediate stageof trophoblasts, which is different from all the existing methods andleads to the following advantages.

Provided herein is a method of selecting clinical grade T-MSC for thetreatment of autoimmune diseases, the T-MSC having the followingcharacteristics: (i) contain >95% of cells expressing group-1 markers;(ii) contain >80% of cells expressing group 2 markers; (iii) contain <5%of cells expressing group-3 markers; (iv) express IL-10 and TGFβ; (v)contain <2% of cells expressing IL-6, IL-12 and TNFα; (vi) express highlevel of CXCR7, CXCL2, CXCL12 but a low level of HOXB2, HOXB3, HOXB5,HOXB7, HOXB9, HOXA5, HOXA9 and other HOX family genes (vii) contain<0.001% of cells co-expressing all group-4 markers, wherein group-1markers are CD73, CD90, CD105, CD146, CD166, and CD44, group-2 markersare CD13, CD29, CD54, CD49E, group-3 markers are CD45, CD34, CD31 andSSEA4, and group-4 markers are OCT4, NANOG, TRA-1-60 and SSEA4.

Provided herein is a method of modifying T-MSC to produce a populationof modified MSC having the following characteristics: (i) contain >95%of cells expressing group-1 markers; (ii) contain >80% of cellsexpressing group 2 markers; (iii) contain <5% of cells expressinggroup-3 markers (iv) expressing IL-10 and TGFβ; (v) contain <2% of cellsexpressing IL-6, IL-12 and TNFα,; and (vi) contains <0.001% of cellsco-expressing all group-4 markers, wherein group-1 markers are CD73,CD90, CD105, CD146, CD166, and CD44, group-2 markers are CD13, CD29,CD54, CD49E, group-3 markers are CD45, CD34, CD31 and SSEA4, and group-4markers are OCT4, NANOG, TRA-1-60 and SSEA4.

Provided herein are conditioned medium, concentrate of conditionedmedium, cell lysate or other derivatives thereof that comprises one ormore biomolecules secreted by the T-MSC as described.

Provided herein is a method of using T-MSC as described herein as feedercells for bone marrow hematopoietic stem cell expansion andumbilical-cord hematopoietic stem cell expansion. In certainembodiments, the T-MSC suitable for the disclosed method express Stro3.In certain embodiments, T-MSC is co-cultured with bone marrowhematopoietic stem cells and/or umbilical-cord hematopoietic stem cells.In certain embodiments, the T-MSC are mesenchymal stromal cells.Provided herein is a co-culture of T-MSC as described herein and bonemarrow hematopoietic stem cells. Provided herein is a co-culture ofT-MSC as described herein and umbilical-cord hematopoietic stem cells.

Also disclosed are kits comprising T-MSC described herein. In certainembodiments, the kits comprise T-MSC and a cell delivery carrier.

In one aspect, provided herein is a method of suppressing or reducing animmune response comprising contacting a plurality of immune cells With aplurality of T-MSC for a time sufficient for the T-MSC, to detectablysuppress an immune response, wherein the T-MSC detectably suppress Tcell proliferation and/or differentiation in a mixed lymphocyte reaction(MLR) assay. In another specific embodiment, the contacting is performedin vitro. In another specific embodiment, the contacting is performed invivo. In a more specific embodiment, the in vivo contacting is performedin a mammalian subject, e.g., a human subject. In another more specificembodiment, the contacting comprises administering the T-MSCintravenously, intramuscularly, or into an organ in the subject (e.g., apancreas).

Provided herein are methods of producing cell populations comprisingT-MSC selected on the basis of their ability to modulate (e.g.,suppress) an immune response. In one embodiment, for example, theinvention provides a method of selecting a T-MSC population comprising(a) assaying a plurality of T-MSC in a mixed lymphocyte reaction (MLR)assay; and (b) selecting the plurality of T-MSC if the plurality ofT-MSC detectably suppresses CD4⁺ or CD8⁺ T cell proliferation in an MLR(mixed lymphocyte reaction), wherein the T-MSC express CD73, CD90,CD105, CD13, CD29, CD54, CD 44. In one embodiment, the T-MSC do notexpress or express at low level CD34, CD31 and CD45. In one embodiment,the T-MSC do not express or express at low level MMP2, RAGE, IFNGR2,IL-12A, IL-6 and VCAM1.

Provided herein are methods to differentiate T-MSC into multiple othercell lineages including, but not limited to, adipocytes, myoblast cells,neural lineage cells. osteoblast cells, fibroblast, chondrocytes, andstromal cells.

Provided herein are methods for using T-MSC and its differentiatedcellular products for tissue regeneration and/or tissue repaircomprising administering T-MSC and/or T-MSC derived other cell lineages,in an amount sufficient to promote tissue regeneration including, butnot limited to, joint regeneration, tendon regeneration, connectivetissue regeneration, neural lineage cells regeneration, fat tissueregeneration, bone regeneration, skin regeneration, muscle regeneration,cartilage regeneration, smooth muscle regeneration, cardiac muscleregeneration, epithelia tissue regeneration, ligament regeneration, etc.

In specific embodiments, the T cells and the T-MSC are present in theMLR at a ratio of, e.g., about 20:1, 15:1, 10:1, 5:1, 2:2, 1:1, 1:2,1:5, 1:10 or 1:20, preferably 10:1.

It is a further object of the disclosed method to efficiently generatelarge numbers of MSCs via a high yield process. The disclosed method cangenerate about 10-fold higher numbers of MSCs compared to the startingnumber of hESCs. There is very little cell loss when hESCs aredifferentiated through the trophoblast stage, whereas, other methodsusually have over 90% loss of the starting cells during the initialdifferentiation step, resulting much lower cell yields than the methoddisclosed herein.

It is an object of the disclosed method to provide a method that canproduce MSCs in a relatively short time. The entire process disclosedherein can be completed in no more than 6-14 days, depending on thestarting hES lines.

It is an object of the disclosed method to provide a method that is lowin cost. The differentiation method described herein only requires avery small amount of culture medium, and the method only requires onecytokine—BMP4, which is used in the disclosed method at a low dose.

It is an object of the disclosed method to provide a method that is lowin cost, The differentiation method described herein only requires avery small amount of culture medium, arid the method only requires onecytokine—BMP4 and/or a TGFβ inhibitor (i.e., SB431542, A83-01 or ALK5inhibitor etc.).

It is an object of the disclosed method to provide a method that is highin yield. The differentiation method described herein can produce1-5×10¹⁰ T-MSC cells within 30 days from 1×10⁵ of hESC, whereas othermethod can only produce up to 1×10⁸ MSC cells within 30 days.

It is a further object of the disclosed method to provide MSCs havinghigh immunosuppressive efficacy. The T-MSC have higher immunosuppressivepotency than MSCs derived from bone marrow (BM) or other sources, theT-MSC have higher immunosuppressive potency than MSCs derived from hESCsvia other method&

In specific embodiments, the T-MSC suppress CD4⁺ or CD8⁺ T cellproliferation by at least 50%, 70%, 90%, or 95% in an MLR compared to anamount of T cell proliferation in the MLR in the absence of the T-MSC.

In another specific embodiment, any of the foregoing compositionscomprises a matrix. In a more specific embodiment, the matrix is athree-dimensional scaffold. In another more specific embodiment, thematrix comprises collagen, gelatin, laminin, fibronectin, pectin,ornithine, or vitronectin. In another more specific embodiment, thematrix is a biomaterial. In another more specific embodiment, the matrixcomprises an extracellular membrane protein. In another more specificembodiment, the matrix comprises a synthetic compound. In another morespecific embodiment, the matrix comprises a bioactive compound. Inanother more specific embodiment, the bioactive compound is a growthfactor, cytokine, antibody, or organic molecule of less than 5,000daltons.

The invention further provides cryopreserved stem cell populations,e.g., a cell population comprising T-MSC, wherein the cell population isimmunomodulatory, which are described herein. For example, the inventionprovides a population of T-MSC that have been identified as detectablysuppressing T cell proliferation and/or differentiation in a mixedlymphocyte reaction (MLR) assay, wherein the cells have beencryopreserved, and wherein the population is contained within acontainer.

In a specific embodiment of any of the foregoing cryopreservedpopulations, the container is a bag. In various specific embodiments,the population comprises about, at least, or at most 1×10⁶ the stemcells, 5×10⁸ the stem cells, 1×10⁷ the stem cells, 5×10⁷ the stem cells,1×10⁸ the stem cells, 5×10⁸ the stem cells, 1×10⁹ the stem cells, 5×10⁹the stem cells, or 1×10¹⁰ the stem cells. In other specific embodimentsof any of the foregoing cryopreserved populations, the stem cells havebeen passaged about, at least, or no more than 5 times, no more than 10times, no more than 15 times, or no more than 20 times. In anotherspecific embodiment of any of the foregoing cryopreserved populations,the stem cells have been expanded within the container.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1 (A-B). (A) Flow chart of the protocol for hESC differentiationinto T-MSCs via a trophoblast and pre-T-MSC stage. Key bio-markers thatare associated with each differentiation stage are indicated. (B)Comparison of various MSC generation protocols for MSC yield andquality: hESCs were differentiated in three protocols. 1) T-MSC: 3 daysin the trophoblast differentiation medium followed by 8-10 days in a MSCgrowth medium. 2) SB MSC: 3-10 days in SB431542-supplementeddifferentiation medium followed by 12 days in the MSC growth medium. 3)HB-MSC: hESC are differentiated into MSC through a hemangioblastintermediate stage, hESC were differentiated into hemangioblast inserum-free medium for 10-13 days followed by 12 days in the MSC growthmedium. The total number of MSCs (millions of cells) in differentcultures at day 10, 20 and 30 following the initiation of thedifferentiation procedures are shown. MSC purity was determined by FACSanalysis of CD73+ cell ratio.

FIGS. 2 (A-C). Morphological changes observed at various time points incultures of hESCs which are in the process of differentiating to T-MSCs.(A) Day 2: trophoblasts; (B) Day 5: pre-MSCs (mesodermal cells); and (C)Day 9: MSCs.

FIGS. 3 (A-C). Analysis of the ratio of cells expressing the trophoblastmarker Trop-2 (Trp-2) and MSC marker CD73 at various time points duringthe differentiation of hESC into T-MSC. (A) Day 2: trophoblasts, (B) Day5: pre-MSCs (mesodermal cells); and (C) Day 9: MSCs.

FIGS. 4 (A-H). Surface. marker expression profile of T-MSC after 11 daysof differentiation. (A) Trp2 is a marker for trophoblasts, (B) CD31 is amarker for endothelial cells, and (C) CD34 is a marker for hematopoieticstem cells. (D-H) CD73, CD90, CD 105, CD44, CD29 are markers for MSCs.

FIGS. 5 (A-R). The in vitro immunosuppressive function of T-MSCs.BM-MSCs (G-L) or T-MSCs (M-R) were mixed with CFSE-labeled mouselymphocytes at 10:1 ratio. The cells were stimulated with anti-CD3antibody at 0.3 or 1 μg/ml together with 1 μg/ml of anti-CD28 antibody.Cell proliferation was indicated by CFSE dilution via FACS analysts.(A-F) T cells cultured without BM-MSC or T-MSC (labeled control) areshown.

FIG. 6. T-MSC attenuate the disease score of an EAE mouse model: EAE wasinduced in C57BL/6 mice with MOG35-55 plus an adjuvant and pertussistoxin. T-MSC, BM-MSC or MSCs derived from hESCs using the SB431542method (hES-MSC(SB)) were intraperitoneously injected into the mice, 6days after the EAE induction. Disease score (from 0 being the no diseaseto 4 being the severe disease) was recorded for 27 days after the MSCinjection.

FIGS. 7 (A-C). Determination of the multipotency of T-MSC todifferentiate into: (A) osteocytes, (B) chondrocytes, and (C)adipocytes.

FIG. 8. Gene expression analysis of comparing hES-HB-MSC (hEShemagioblast derived MSC) with T-MSC (hES trophoblast derived MSC) andBM-MSC (adult bone marrow derived MSC). Gene expression was normalizedand is shown as arbitrary expression units.

DETAILED DESCRIPTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the methods of the invention and howto use them. Moreover, it will be appreciated that the same thing can bethe in more than one way. Consequently, alternative language andsynonyms may be used for any one or more of the terms discussed herein,nor is any special significance to be placed upon whether or not a termis elaborated or discussed herein. Synonyms for certain terms areprovided. A recital of one or more synonyms does not exclude the use ofthe other synonyms, The use of examples anywhere in the specification,including examples of any terms discussed herein, is illustrative only,and in no way limits the scope and meaning of the invention or anyexemplified term. Likewise, the invention is not limited to itspreferred embodiments.

The term hESC means human embryonic stem cells that encompasspluripotent stem cells produced from embryo, inner cell mass, blastomereor a cell line.

The term “hES-MSC” or hES-MSCs” or “human embryonic mesenchymal stemcells” or human embryonic stem cell derived mesenchymal stem cells” or“hES-MSC population” as used herein means mesenchymal-like stem cells,mesenchymal like stromal cells, mesenchymal stem cells or mesenchymalstromal cells, derived from human embryonic stem cells or derived frominduced pluripotent stem cells (“iPSCs”) using any methods. hES-MSC asused herein includes individual cells, cell lines, batches, lots orpopulations of hES-MSC.

The term “T-MSC” refers to MSC or mesenchymal stem/stromal cells thatare derived from human embryonic stem cells (hESC) or inducedpluripotent stem cells (iPSC) through a trophoblast intermediate stagewhere cells express Trop-2 with trophoblast-like morphology. The term“hES-T-MSC” refers to T-MSC differentiated from hESC. The term“iPS-T-MSC” and “iT-MSC” refer to T-MSC differentiated from iPSC. Theterm “T-MSC” as used herein does not refer to a trophoblast. A cell isconsidered a “stem cell” if the cell retains at least one attribute of astem cell, e.g., the ability to differentiate into at least one othertype of cell, or the like. These cells can be described based uponnumerous structural and functional properties including but not limitedto, expression or lack of expression of one or more markers. T-MSCs,including both hES-T-MSC and iT-MSC, are multipotent and capable ofdifferentiating to give rise to other cell types and cell lineages.

The term “hES-HB-MSC” and “HB-MSC” are mesenchymal stem cells that arederived from human pluripotent stem cells including hESC and iPSCs viahemangioblast or hemangio-colony forming middle step.

The term “clinical grade T-MSC” as used herein means T-MSC whichcontains characteristics that are suitable for use in clinical use forhuman, avian or other mammals. Clinical grade T-MSC as used hereinincludes individual cells, cell lines, batches, lots or populations ofMSC.

The term “T-MSC population” as used herein means a population of T-MSCcells which contains cells that have characteristics that are suitablefor use in treatment and cells that do not have characteristics that aresuitable for use in treatment

The term “T-MSC derived lineages” or T-MSC-DL as used herein means cellsor cell lineages differentiated from T-MSC including, but not limitedto, adipocytes, myoblast cells, neural lineage cells, osteoblast cells,fibroblast, chondrocytes, and stromal cells.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to cause an improvement in a clinically significantcondition in the subject, or delays or minimizes or mitigates one ormore symptoms associated with the disease, or results in a desiredbeneficial change of physiology in the subject.

The terms “treatment”, and the like refer to a means to slow down,relieve, ameliorate or alleviate at least one of the symptoms of thedisease, or reverse the disease after its onset.

The terms “prevent”, “prevention”, and the like refer to acting prior toovert disease onset, to prevent the disease from developing or minimizethe extent of the disease or slow its course of development.

The term “subject” as used in this application means an animal with animmune system such as avians and mammals. Mammals include canines,felines, rodents, bovine, equines, porcines, ovines, and primates.Avians include, but are not limited to, fowls, songbirds, and raptors.Thus, the invention can be used in veterinary medicine, e.g., to treatcompanion animals, farm animals, laboratory animals in zoological parks,and animals in the wild. The invention is particularly desirable forhuman medical applications

The term “in need thereof” would be a subject known or suspected ofhaving or being at risk of developing a disease including but notlimited to multiple sclerosis and other T cell related autoimmunediseases, or diseases related to the central nervous system or theblood-brain barrier or the blood-spinal cord barrier.

A subject in need of treatment would be one that has already developedthe disease. A subject in need of prevention would be one with riskfactors of the disease.

The term “agent” as used herein means a substance that produces or iscapable of producing an effect and would include, but is not limited to,chemicals, pharmaceuticals, drugs, biologics, small molecules,antibodies, nucleic acids, peptides, and proteins.

As used herein, a stem cell is “positive” for a particular marker whenthat marker is detectable. For example, a T-MSC is positive for, e.g.,CD73 because CD73 is detectable on T-MSC in an amount detectably greaterthan background (in comparison to, e.g., an isotype control). A cell isalso positive for a marker when that marker can be used to distinguishthe cell from at least one other cell type, or can be used to select orisolate the cell when present or expressed by the cell.

As used herein, “immunomodulation” and “immunomodulatory” mean causing,or having the capacity to cause, a detectable change in an immuneresponse, and the ability to cause a detectable change in an immuneresponse.

As used herein, “immunosuppression” and “immunosuppressive” meancausing, or having the capacity to cause, a detectable reduction in animmune response, and the ability to cause a detectable suppression of animmune response.

The present invention is based on the first discovery that mesenchymalstem cells MSCs can be differentiated from the hESC derivedtrophoblasts, and that the trophoblast-derived MSCs (T-MSC) can be usedfor tissue repair and immune regulation. These T-MSC produced from thedisclosed methods all remarkably inhibited T cell proliferation anddifferentiation in vitro and attenuated the disease score in vivo,whereas bone marrow-derived MSC (BM-MSC) had no effect at all in vivo,although the BM-MSC may partially reduce T cell proliferation anddifferentiation in vitro. The T-MSC disclosed herein have surprisinglyhigher immunosuppressive activity compared to BM-MSC. The methodsdisclosed herein are highly efficient and can produce high number ofT-MSC with low cost and high purity. The methods disclosed herein arehighly reproducible with little batch-to-batch variations, and easilyadaptable to meet clinical needs.

Thus, the present invention overcomes the problems described above byproviding a method of generating mesenchymal stem cells (MSC) in vitrofrom human embryonic stem cells. The ability to generate the hES-T-MSCby the methods disclosed herein allows the production of cells that canbe used in a variety of therapeutic applications, including thetreatment and prevention of multiple sclerosis, and other autoimmunediseases. Additionally, the hES-MSC produced by the methods describedherein have the ability to cross the brain-blood barrier (BBB) and theblood-spinal cord barrier (BSCB) allowing them to be used for a varietyof therapeutic applications, including drug delivery. The methods of theinvention provide further utility in that they enable the generation oflarge numbers of hES-T-MSC that can be used on a commercial scale.

Differentiation of Embryonic Stem Cells through Trophoblast to ObtainT-MSC

Disclosed herein is a method for generating and expandingmesenchymal-like stem cells (MSCs) from trophoblast derived fromembryonic stem cells (hES). These resulting cells are designated T-MSC.These T-MSG can be isolated and/or purified.

MSC-like cells have been derived from human embryonic stem cells byvarious methods (Barbieri et al. (2005); Olivier et al. (2006); Sanchezet al. (2011); Brown et al. (2009)). However, all of these methodsinvolve co-culturing and hand-picking procedures that limit yield andpurity and result in varying quality of cells.

Although hESC express low levels of MHC antigens, it has been found thatmany cell types differentiated from hESC have increased expression ofthese antigens (Draper et al., 2002; Drukker et al., 2006; Drukker etal., 2002), thus, causing great concern for immunorejection of thedifferentiated cells if transplanted into patients. In contrast, MSCexpress low levels of costimulatory molecules and major MHC antigens,and have been used in allogeneic or xenograft models to treat autoimmunediseases (Gordon et al., 2008b, Grinnemo et al., 2004; Rafei et al.,2009a; Rafei et al., 2009b; Tse et al., 2003). T-MSC, like adulttissue-derived MSC, express low levels of the co-stimulatory moleculesand MHC antigens, and do not require long-term engraftment to exertimmunosuppressive effect, thus, there is no concern for immunorejectiondue to mismatch of MHC antigens between MSC and the recipient. One hESCline is sufficient to generate T-MSC at large scale, in an endlesssupply, and with easy quality control, suitable for industrialproduction as a potential therapy to treat patients with MS and other Tcell-based autoimmune diseases.

Human trophoblast can be generated from human embryonic stem cells. Suchembryonic stem cells include embryonic stem cells derived from or using,for example, blastocysts, plated ICMs, one or more blastomeres, or otherportions of a pre-implantation-stage embryo or embryo-like structure,regardless of whether produced by fertilization, somatic cell nucleartransfer (SCNT), parthenogenesis, androgenesis, or other sexual ofasexual means.

Additionally or alternatively, trophoblast can be generated from otherembryo-derived cells. For example, trophoblast can be generated (withoutnecessarily going through a step of embryonic stem cell derivation) fromor using plated embryos, ICMs, blastocysts, one or more blastomeres,trophoblast stem cells, embryonic gear cells, or other portions of apre-implantation-stage embryo or embryo-like structure, regardless ofwhether produced by fertilization, somatic cell nuclear transfer (SCNT),parthenogenesis, androgenesis, or other sexual or asexual means.Similarly, trophoblast can be generated using cells or cell linespartially differentiated from embryo-derived cells. For example, if ahuman embryonic stem cell line is used to produce cells that are moredevelopmentally primitive than trophoblast, in terms of developmentpotential and plasticity, such embryo-derived cells could then be usedto generate trophoblast.

Additionally or alternatively, trophoblast can be generated from otherpre-natal or peri-natal sources including, without limitation, umbilicalcord, umbilical cord blood, amniotic fluid, amniotic stem cells, andplacenta.

The human embryonic stem cells may be the starting material of thismethod. The embryonic stem cells may be cultured in any way known in theart, such as in the presence or absence of feeder cells.

In the examples set forth herein, eight hESC cell lines were used, H9(derived from WiCell Research Institute) (Thomson et al. (1998), CT2(derived from University of Connecticut Stem Cell Core (Lin et al.(2010)); and ES03-Envy (Envy, a GFP-labeled line, derived at ESInternational) (Costa et al. (2005)), ESI-017, ESI-053, ESI-049,ESI-035, and ESI-051.

In the first step of this method to obtain T-MSC, human embryonic stemcells are grown in small clumps or single cells in serum-free mediawithout bFGF. The cells are then re-plated and cultured with BMP4 (1-200ng/ml) as the only cytokine for a short time (2-5 days) to obtain ahighly homogenous population of trophoblasts as they express the typicaltrophoblast marker Trop2/TACSTD2 (Trp2). A TGFβ inhibitor (SB431542(1-20 μM), A83-01(0.2-5 μM) or ALK5 inhibitor (1-20 μM), etc.) can beused to increase the trophoblast forming efficiency. The cells willexpand and differentiate into trophoblast cells in 2-5 days withtrophoblast-like morphology, in certain embodiments, more than 90% ofcells express Trop-2/TACSTD2 (Trp-2) (Xu et al., 2002). Trophoblasts maybe isolated by size or purified with antibody, such as by immunoaffinitycolumn chromatography.

In one embodiment, trophoblast cells are digested to form single cellswith TrypLE, Trypsin or collagenase B. The single cells are re-suspendedin a medium optimized for mesenchymal stem cell growth such as alpha-MEMcontaining 2-20% of fetal bovine serum (FBS) or human AB serum (ABHS),DMEM-high glucose containing 2-20% of FBS or ABHS, the FBS can bereplaced with 5-20% of knock-out serum replacement (KOSR) or bovineserum albumin (BSA), or any other commercial available serum free MSCculture mediums. In certain embodiments, Serum, KOSR or BSA is added ina concentration of from about 5-20%. In certain embodiments, fetalbovine serum is preferred. In certain embodiments, cells are cultured ata density of about 10-1000 cells/cm². In certain embodiments, the cellsare cultured in an environment that mimics the extracellular environmentof tissues, such as gelatin, vitronectin, laminin, fibronectin, collagenI. In certain embodiments, the MSC culture medium comprises LIF (2-20ng/ml), bFGF (2-100 ng/ml), or PDGF (1-50 ng/ml) to increase expansionefficiency.

After approximately 24 hours, a number of cells (50-90%) attached to theculture plate and approximately 2-3 days later, pre-T-MSC begin todifferentiate from the trophoblasts, cells were elongated and form clearcell border. In certain embodiments, the pre-T-MSC express both CD73 andTrop-2. After 6-10 days, more than 80-90% cells trophoblasts aredifferentiated into mesenchymal-like small cell with spindle-likemorphology, so called T-MSC here. T-MSC can also be identified by theexpression of certain markers, such as CD73, CD90, CD105, CD13, CD29,CD54, CD44, CD146 and CD166 and by the absence or low expression ofcertain markers such as CD31, CD34, and CD45. In certain embodiments,T-MSC do not express HOX and HLA-G. In certain embodiments, T-MSCexpress high level of CXCR7, CXCL2, CXCL12 but low level of HOXB2,HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and other HOX family genes.T-MSC are also characterized as multipotent and able to differentiateinto adipocytes chondrocytes, osteoblast cells, neurons, myoblasts,stromal cells and fibroblasts.

Provided herein is an isolated cell population comprising a plurality ofimmunosuppressive T-MSC that expresses at least one of the followingmarkers: CD73, CD90 and CD0105.

In a further embodiment of the present invention, an additional step ofirradiating the T-MSCs is performed. This irradiation can beaccomplished with the use of any method known in the art that emitsradiation including but not limited to gamma irradiation e.g.,Cesium-137 gamma irradiation, or photon radiation using X-ray. Thepreferred amount of radiation to be administered is about between 5 and20000 gy, more preferably about between 50 and 100 gy, and mostpreferably 80 gy.

In one embodiment, the method described herein is a novel process forderiving (also referred to herein as producing) T-MSC from hESCs. Themethod comprises the steps of:

-   -   a. Culturing a cell culture comprising human embryonic stem        cells in serum-free medium in the present of at least one growth        factor in an amount sufficient to induce the differentiation of        the embryonic stem cells to differentiate into trophoblast; in        an embodiment, the time period of the differentiation into        trophoblast is about 2-5 days; in an embodiment, the medium        comprises BMP4, with or without the presence of an TGFb        inhibitor (i.e., SB431542, A83-01 or ALK5 inhibitor etc.) to        increase the differentiation efficiency:    -   b. Adding at least one growth factor to the culture comprising        the trophoblasts and continuing to culture in serum-free medium,        wherein the growth factor is in an amount sufficient to expand        the trophoblasts, in an embodiment, the medium comprises BMP4,        (this step is optional);    -   c. Isolating the trophoblasts and re-plating the trophoblasts        onto gelatin, laminin, fibronectin, vitronectin, collagen or        Matrigel-coated plates and cultured in a serum-containing or        serum-free media in an amount sufficient to differentiate the        trophoblast into T-MSC through pre-T-MSC, in an embodiment, the        isolated trophoblast is cultured for 6-10 days to produce the        T-MSC, wherein at least about 90%, 95%, 96%, 97%, 98%, 99% of        the resulting T-MSC express cell surface markers for adult MSCs,        in an embodiment, the medium comprises LIF, bFGF, PDGF to        increase expansion efficiency,        wherein at least about 90%, 95%, 96%, 97%, 98%, 99% of the        resulting T-MSC express cell surface markers for adult MSCs.

As shown in FIGS. 1 & 2, the disclosed method starts with dispersal ofhESC colonies into small clumps or single cells. The cells are thenre-plated and cultured with BMP4 as the only cytokine, and a TGFβinhibitor for a short time (2-5 days) to obtain a highly homogenouspopulation of trophoblasts as they express the typical trophoblastmarker Trop-2/TACSTD2 (Trp-2) (Xu et al., 2002). The trophoblasts arethen dissociated and re-plated onto a gelatin, laminin, fibronectin,vitronectin, collagen or matrigel-coated plate and cultured in a MSCgrowth medium for 4-10 days to generate spindle-like cells similar tothe morphology of typical MSCs.

The method disclosed herein, unlike the other methods, does not requirefeeder cells, sorting or hand-picking of the cells. The initialtrophoblast differentiation step is in a defined, serum-free mediumwithout bFGF. The entire protocol only requires two steps ofdifferentiation in a total of 6-14 days to generate T-MSC at high purityand high yield (FIG. 1). This is the shortest differentiation protocolever reported for MSC derivation from hESC. The yield and purity of theT-MSC are very high compared to those achieved using previously reportedmethods. Within 30 days, T-MSC at 5×10⁵ fold the number of the originalhESCs can be obtained and with a high percentage of CD73+ cells, atypical marker for MSCs, whereas the other methods can only yield lessthan 100 fold the original hESC number with a low percentage of CD73+cells. The derivation of the T-MSC includes an intermediate stage ofCD73/Trp-2 double positive cells, hereafter named pre-T-MSC. After 2-3,days of the BMP4 plus a TGFβ inhibitor treatment, the cells firstexpress Trp-2 at a high percentage and demonstrate a homogenousmorphology of trophoblasts (FIGS. 2 & 3). After 5-6 days, the cellsexpress both Trp-2 and CD73; after 6-14 days, the cells no longerexpress Trp2 but express the typical MSC surface markers at highpercentages including CD73 (>98%), CD90 (>95%), CD105 (>90%), CD44(>95%), CD29(>80%); and the cells are negative for the endothelialmarker CD31 and hematopoiesis markers CD34 and CD45 (FIGS. 3 & 4).

T-MSC produced by the method disclosed herein are capable ofdifferentiating to downstream osteogenesis, chondrogenesis andadipogenesis lineages (FIG. 7). Thus, the T-MSC are phenotypically andfunctionally similar to MSCs derived from the bone marrow (BM) and othersources.

Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells

Bone marrow-derived MSCs (BM-MSCs) have long been used to treatautoimmune disease in many animal models and clinical trials, howeverthe efficacy of immunosuppression is not consistent with some reportsshowing BM-MSCs are unable to efficiently treat certain autoimmunediseases (Tyndall, 2011). Data is provided herein comparing the abilityof BM-MSCs and T-MSC for their inhibition of T cell proliferationfollowing T cell receptor stimulation. As shown in FIG. 7, BM-MSCs caninhibit proliferation of both CD4 and CD8 T cells induced by anti-CD3antibody at a low dose (0.3 ug/ml), which is comparable to T-MSC.However, when the anti-CD3 antibody concentration increased to 1 ug/ml,BM-MSCs have less potency in suppressing proliferation of both CD4 andCD8 T cells than T-MSC. CFSE dilution assay was used here to evaluatethe T cell proliferation: an increased percentage of T cells withdecreased CFSE signal indicates an accelerated proliferation. As shownin FIG. 5, when anti-CD3 antibody increased to 1 ug/ml, there were 59%of CD4 and 46% of CD8 T cells detected with decreased CFSE signal. T-MSCsignificantly decreased both the CD4 and CD8 T cells to 16%, whereasBM-MSCs only decreased CD4 and CD8 T cells to 32% and 36%, respectively.

Consistent with the in vitro immunosuppressive activity of the T-MSC.T-MSC produced by the method disclosed herein were shown to be effectiveto treat experimental autoimmune encephalomyelitis (EAE), a mouse modelof multiple sclerosis. As shown in FIG. 6, when T-MSC were injected 6days post the EAE induction, the disease score of the EAE micesignificantly declined, compared to vehicle injection controls.

In a further feature of cells produced by the disclosed methods, T-MSCalso demonstrated much stronger immunosuppressive effect than BM-MSCsand hES-MSCs derived through SB431542 treatment (Chen et al., 2012)(FIG. 6). In several repeated experiments, BM-MSCs consistently failedto attenuate the disease score of EAE mice. Thus, the replacement ofBM-MSCs with T-MSC produced by the disclosed method for use in clinicalapplications would remove the need for risky, invasive procedures forbone marrow aspiration, reduce the time for waiting for BM donations,reduce the cost, and reduce batch to batch variations for preparingBM-MSCs on a per-patient basis.

In summary, disclosed herein is a highly efficient method to generatemesenchymal-like cells or MSCs from hESCs through an intermediatetrophoblast stage, and the use of the T-MSC to treat autoimmune disease.Microarray analysis suggested that the T-MSC had a gene expressionprofile not identical to that of BM-MSCs (data not shown), although bothcan differentiate into the same downstream cell lineages (FIG. 7). Inaddition, the T-MSC have stronger immunosuppressive ability both invitro and in vivo than BM-MSCs.

The available data suggest that T-MSC produced by the disclosed methodare different from traditional, adult-derived MSCs. Due to their stronginhibition of T cell proliferation, T-MSC may be used to treat multiplesclerosis with much higher efficacy than BM-MSCs. To address potentialsafety concerns, T-MSC were injected into immunodeficient SCID-beigemice. No tumor or teratoma formation was observed in the mice.

The T-MSC of the present invention are unique and have a variety oftherapeutic and other uses. Thus, the present invention includes variouspreparations, including pharmaceutical preparations, and compositionscomprising T-MSC.

The term “T-MSC” refers to MSC or mesenchymal stem/stromal cells thatare derived from human embryonic stem cells (hESC) or inducedpluripotent stem cells (iPSC) through a trophoblast intermediate stagewhere cells express Trop-2 with trophoblast-like morphology. The term“hES-T-MSC” refers to T-MSC differentiated from hESC. The term“iPS-T-MSC” and “iT-MSC” refer to T-MSC differentiated from iPSC. Theterm “T-MSC” as used herein does not refer to a trophoblast. A cell isconsidered a “stem cell” if the cell retains at least one attribute of astem cell, e.g., the ability to differentiate into at least one othertype of cell, or the like. These cells can be described based uponnumerous structural and functional properties including but not limitedto, expression or lack of expression of one or more markers.Specifically, T-MSC are characterized by small cell bodies with afibroblast morphology. T-MSCs, including both hES-T-MSC and iT-MSC, aremultipotent and capable of differentiating to give rise to other celltypes and cell lineages. The term “T-MSC-DL” refers to all the celltypes and cell lineages differentiated from T-MSC.

The differentiation. method described herein can achieve thedifferentiation of MSC from iPS cells within 6-14 days, the shortesttime ever reported. Thus, these iT-MSC can be used for patient specificiPS based therapy under emergency conditions which requires thegeneration of MSC in very short time, such as acute heart infarction,acute heart failure, acute spinal cord injury, acute radiation/burningtreatments, etc.

T-MSC can be identified or characterized by the expression or lack ofexpression as assessed on the level of DNA, RNA or protein, of one ormore cell markers. T-MSC can be identified as expressing cell surfacemarker CD73, or expressing at least one or more of the following cellsurface markers: CD90, CD105, CD13, CD29, CD54, CD44, CD146 or CD166 ornot expressing or expressing at a low level at least one of thefollowing cell surface markers: CD34, CD31, or CD45.

Alternatively or additionally, T-MSC can be identified or characterizedbased upon their low level of expression of one or more pro inflammatoryproteins, MMP2, RAGE, IFNGR2, TNFα, IL-12A, IL-6, and VCAM1. Thisprofile of gene expression is in contrast to bone marrow derivedmesenchymal stem cells. In particular, IL-6 was expressed much higher inBM-MSCs than in T-MSC. IL-6 is a pleiotropic cytokine involved incrosstalk between hematopoietic/immune cells and stromal cells,including the onset and resolution of inflammation.

The T-MSC can also be characterized in their ability to inhibit T cellproliferation after stimulation in vitro. This characteristic is incontrast to BM-MSCs which do not inhibit T cell proliferation afterstimulation in vitro.

Thus, the T-MSC described herein have at least one of the followingcharacteristics: (1) differentiate into adipocytes, chondrocytes,osteoblast cells, neurons, myoblasts, stromal cells and fibroblasts; (2)have a fibroblast-like morphology; (3) express CD73, CD90, CD105, CD13,CD29, CD54, CD44, CD146 and/or CD166; (4) express at low levels or donot express CD34, CD31, and/or CD45; (5) express at low levels or do notexpress MMP2, RAGE, IFNγR1, IFNγR2, IL-12, TNFα, IL-6, and/or VCAM1,particularly IL-6; (6) express MHC antigen HLA-G and/or HLA-ABC andexpress at low levels or do not express HLA-DR and/or CD80; and (7)inhibit T cell proliferation after stimulation in vitro. In certainembodiments, the T-MSCs have at least two, at least three, at leastfour, at least five, at least six, or all seven characteristics.

In certain embodiments, T-MSC is distinguishable with previouslyreported HB-MSC, T-MSC express at least one fold higher level of CXCR7,CXCL2 and/or CXCL12 than HB-MSC, but at least half of the level ofHOXB2, HOXB3, HOXB5, HOXB7, HOXB9, HOXA5, HOXA9 and other HOX familygenes compared to HB-MSC.

Additionally, the T-MSC have the unique ability to cross the blood-brainbarrier (BBB) and the blood-spinal cord barrier (BSCB), making themuniquely suited for therapeutic and diagnostic applications. The T-MSCof the current invention have the ability to migrate in and out of thevessels of the spinal cord, across the BSCB, to fulfill functions in theCNS, including but not limited to the delivery of therapeutic anddiagnostic agents. This is in contrast to BM-MSCs which do not have thisability.

Another embodiment of the present invention is a T-MSC that isirradiated. This embodiment would include T-MSC with at least one of thefollowing characteristics listed above, having at least two, at leastthree, at least four, at least five, at least six, or all sevencharacteristics that have been subject to irradiation.

In another embodiment, the cell culture comprises T-MSC. In certainembodiments, the T-MSC differentiate into adipocytes, chondrocytes,osteoblast cells, neurons, myoblasts, stromal cells and fibroblasts. Incertain embodiments. the T-MSC cells express CD73, CD90, CD105, CD13,CD29, CD54, CD44, CD146, and/or CD166. In certain embodiments, the cellsexpress at tow levels or do not express CD34, CD31, anchor CD45. Incertain other embodiments, the cells express at low levels or do notexpress MMP2, RAGE. IFNγR1, IFNγR2, IL-12. TNFα, IL-6, and/or VCAM1,especially IL-6. In certain other embodiments, the cells express MHCantigen HLA-G and/or HLA-ABC and express at low levels or do not expressHLA-DR and/or CD80. In certain other embodiments, the cells inhibit Tcell proliferation after stimulation in vitro. In certain embodiments,the cells can cross the blood-brain harrier and the blood-spinal cordbarrier. In certain embodiments, the cells have been irradiated.

In another aspect, disclosed herein a pharmaceutical preparationcomprising T-MSC. In certain embodiments, the T-MSC can differentiateinto adipocytes, chondrocytes, osteoblast cells, neurons, myoblasts,stromal cells and fibroblasts. In certain embodiments, the cells expressCD73, CD90, CD105, CD13, CD29, CD54, CD44, CD146 and/or CD166. Incertain embodiments, the cells express at low levels or do not expressCD34, CD31, and/or CD45. In certain other embodiments, the cells expressat low levels or do not express MMP2, RAGE, IFNγR1, IFNγR2, TNFα, IL-12,IL-6, and/or VCAM1, especially IL-6. In certain other embodiments, thecells express MHC antigen HLA-G and/or HLA-ABC and express at low levelsor do not express HLA-DR and/or CD80. In certain other embodiments, thecells inhibit T cell proliferation after stimulation in vitro. Incertain embodiments, the cells can cross the blood-brain barrier and theblood-spinal cord barrier. In certain embodiments, the cells have beenirradiated. The pharmaceutical preparation can be prepared using anypharmaceutically acceptable carrier or excipient.

In certain embodiments, the composition or pharmaceutical preparationcomprises at least at least 10,000 T-MSC, at least 50,000 T-MSC, atleast 100,000 T-MSC, at least 500,000 T-MSC, at least 1×10⁵ T-MSC, atleast 5×10⁶ T-MSC, at least 1×10⁷ T-MSC, at least 5×10⁷ T-MSC, at least1×10⁸ T-MSC, at least 5×10⁸ T-MSC, at least 1×10⁹ T-MSC, at least 5×10⁹T-MSC, or at least 1×10¹⁰ T-MSC.

Provided herein are pluralities of T-MSC that comprise T-MSC obtainedand isolated directly from a human embryonic stem cell line that havebeen cultured and passaged at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,14, 16, 18, 20, 25, 30 or more times, or a combination thereof.

In certain embodiments, provided herein is a cryopreserved preparationof T-MSC or cells partially or terminally differentiated therefrom.

In certain embodiments, provided herein is a therapeutic use of T-MSC,or compositions or preparations of T-MSC, including irradiated T-MSC.Such cells and preparations can be used in the treatment of any of theconditions or diseases as described, as well as in a delivery system foragents across the blood-brain barrier and the blood-spinal cord barrier.

In certain embodiments, the invention provides a cryopreservedpreparation of trophoblasts, pre-T-MSC, or T-MSC cells partially orterminally differentiated therefrom.

In certain embodiments, the invention provides the therapeutic use ofT-MSCs, or compositions or preparations of T-MSCs, including irradiatedT-MSCs. Such cells and preparations can be used in the treatment of anyof the conditions or diseases detailed throughout the specification, aswell as in a delivery system for agents across the blood-brain barrierand the blood-spinal cord barrier.

Selecting and Producing T-MSC Populations

Provided herein is a method of identifying highly immunosuppressiveT-MSC by identifying a biomarker profile of the highly immunosuppressiveT-MSC that are clinical grade for use in therapy. In certainembodiments, the clinical grade T-MSC have the followingcharacteristics: (i) contain >95% of cells expressing group-1 markers;(ii) contain >80% of cells expressing group 2 markers; (iii) contain <5%of cells expressing group-3 markers (iv) express IL-10 and TGFβ; (v)contain <2% of cells expressing IL-6, IL-12 and TNFα; and (vi) contains<0.001% of cells co-expressing all group-4 markers, wherein group-1markers are CD73, CD90, CD105, CD146, CD166, and CD44, group-2 markersare CD13, CD29, CD54, CD49E, group-3 markers are CD45, CD34, CD31 andSSEA4, and group-4 markers are OCT4, NANOG, TRA-1-60 and SSEA4.

In certain embodiments, the method comprises measuring the differentialexpression of markers that encode anti-inflammatory factors (“AIF”) andpro-inflammatory factors (“PIF”). In certain embodiments, the AIF isIL-10, TGFβ2. In certain embodiments, the PIF is up regulated. Incertain embodiments, T-MSC express at least 1.5 fold of the abovemarkers as compared to BM-MSC. In certain embodiments, the PIF is IL-6,IL-12, TNFα, CCL2, VCAM1, RAGE, MMP2. In certain embodiments, the PIF isdown regulated. In certain embodiments, T-MSC express at least half ofthe above markers as compared to BM-MSC In another embodiment, highlyimmunosuppressive T-MSC has a lower ratio of IL-6⁺ cells as compared toBM-MSC. In certain embodiments, highly immunosuppressive T-MSC have lessthan 5%, 4%, 3%, 2%, or 1% of IL-6 positive cells. In certainembodiments, T-MSC express low levels of IL12, TNFα, RAGE and other PIF.In certain embodiments, T-MSC may express nigh levels of TGFβ2 andIL-10. In certain embodiments, the expression of markers is compared toexpression in BM-MSC.

Provided herein is a qualification procedure for clinical grade T-MSCpopulation. Expression of specific markers is measured in a populationof T-MSC to determine whether they are suitable for therapeutic use. Themarkers include, for example, (1) MSC-specific markers (set 1): CD73,CD90, CD105, CD188, and CD44, (2) MSC-specific markers (set 2): CD13,CD29, CD54, CD49E, SCA-1, and STRO-1, (3) hernatopoietic stem/progenitormarkers: CD45 and CD34, and endothelial cell marker CD31, (4)immunogenic markers: HLA-ABC, HLA-G, CD80, and CD86, (5) cytokines:IL-10, TGFβ, IL-6, and IL-12, and (6) pluripotency markers: OCT4, NANOG,TRA-1-60, and SSEA-4. In certain embodiments, T-MSC population containsmore than 95%, 96%, 97%, 98%, or 99% of cells that express at least onegroup 1 markers. In certain embodiments, T-MSC population contains morethan 80%, 85%, 90%, 95%, or 99% of cells that express at least one group2 markers. In certain embodiments, T-MSC population contains less than0.1%, 0.08%, 0.05%, 0.03%, 0.02%, or 0.01% of cells that express atleast one group 3 marker. In certain embodiments, T-MSC populationcontains more than 80%, 85%, 90%, 95%, or 99% of cells that expressIL-10 and/or TGFβ. In certain embodiments, T-MSC population containsless than 5%, 4%, 3%, 2%, 1% of cells that express IL-6 and/or IL-12. Incertain embodiments, T-MSC population contains less 0.001% of cells thatexpress at least one group 6 marker. The clinical-grade T-MSC iscompared with the preclinical-grade T-MSC as a positive control. Incertain embodiments, the T-MSC is characterized through multi-color flowcytometry analyses and/or immunofluorescence. In certain embodiments,T-MSC population express CCL2, CCL3, CCL4, CCL5, IL-1, IL-2, IL-4, IL-6,IL-8, IL-10, IL-17, TNFα, TGFβ, IFNγ, GM-CSF, G-CSF, bFGF, CXCL5, VEGF,TPO or a combination thereof. In certain embodiments, the T-MSCpopulation will also be analyzed for (1) presence of exogenous materialssuch as endotoxin and residual cytokines/growth factors. and/or (2)genomic abnormalities (via karyotyping and whole-genome sequencing).

Provided herein is another qualification procedure for clinical gradeT-MSC population. T-MSC with better regeneration potential andimmunosuppressive function may express a lower level of CD9, where CD9expression level of Passage 1-2 T-MSC will be recorded as basal level,if after certain passages and procedures, the CD9 expression levelincreases by 2 fold, the cells will be stopped for passaging.

Methods for determining the expression profile of the T-MSC are known inthe art, including but not limited to, flow cytometry, multiplexmicroarray, RT-PCT, Northern blot and Western blot. In certainembodiments, the expression profile of the MSC are determined bycytometric bead array based multiplex cytokine analysis, luminex systembased multiplex cytokine analysis, microarray RNA-seq, quantitativeRT-PCR, Elispot Elisa, Elisa cytokine array, flow cytometry luciferasereporter system, fluorescence reporter system, histology staining, andimmunofluorescence staining.

Methods of Detecting Nucleic Acid Biomarkers

In specific embodiments, biomarkers in a biomarker profile are nucleicacids. Such biomarkers and corresponding features of the biomarkerprofile may be generated, for example, by detecting the expressionproduct (e.g., a polynucleotide or polypeptide) of one or more markers.In a specific embodiment, the biomarkers and corresponding features in abiomarker profile are obtained by detecting and/or analyzing one or morenucleic acids expressed from a marker disclosed herein using any methodwell known to those skilled in the art including, but not limited to,hybridization, microarray analysis, RT-PCR, nuclease protection assaysand Northern blot analysis.

In certain embodiments, nucleic acids detected and/or analyzed by themethods and compositions of the invention include RNA molecules such as,for example. expressed RNA molecules which include messenger RNA (mRNA)molecules, mRNA spliced variants as well as regulatory RNA, cRNAmolecules (e.g., RNA molecules prepared from cDNA molecules that aretranscribed in vitro) and discriminating fragments thereof.

In specific embodiments, the nucleic acids are prepared in vitro fromnucleic acids present in, or isolated or partially isolated from a cellculture, which are well known in the art, and are described generally,e.g., in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual.3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y.),which is hereby incorporated by reference in its entirety.

Nucleic Acid Arrays

In certain embodiments, nucleic acid arrays are employed to generatefeatures of biomarkers in a biomarker profile by detecting theexpression of any one or more of the markers described herein. In oneembodiment of the invention, a microarray such as a cDNA microarray isused to determine feature values of biomarkers in a biomarker profile.Exemplary methods for cDNA microarray analysis are described below, andin the examples.

In certain embodiments, the feature values for biomarkers in a biomarkerprofile are obtained by hybridizing to the array delectably labelednucleic acids representing or corresponding to the nucleic acidsequences in mRNA transcripts present in a biological sample (e.g.,fluorescently labeled cDNA synthesized from the sample) to a microarraycomprising one or more probe spots.

Nucleic acid arrays, for example, microarrays, can be made in a numberof ways, of which several are described herein below. Preferably, thearrays are reproducible, allowing multiple copies of a given array to beproduced and results from the microarrays compared with each other.Preferably, the arrays are made from materials that are stable underbinding (e.g., nucleic acid hybridization) conditions. Those skilled inthe art will know of suitable supports, substrates or carriers forhybridizing test probes to probe spots on an array, or will be able toascertain the same by use of routine experimentation.

Arrays, for example, microarrays, used can include one or more testprobes. In some embodiments, each such test probe comprises a nucleicacid sequence that is complementary to a subsequence of RNA or DNA to bedetected. Each probe typically has a different nucleic acid sequence,and the position of each probe on the solid surface of the array isusually known or can be determined. Arrays useful in accordance with theinvention can include, for example, oligonucleotide microarrays, cDNAbased arrays, SNP arrays, spliced variant arrays and any other arrayable to provide a qualitative, quantitate or semi-quantitativemeasurement of expression of a marker described herein. Some types ofmicroarrays are addressable arrays. More specifically, some microarraysare positionally addressable arrays. In some embodiments, each probe ofthe array is located at a known, predetermined position on the solidsupport so that the identity (e.g., the sequence) of each probe can bedetermined from its position on the array (e.g., on the support orsurface). In some embodiments, the arrays are ordered arrays.Microarrays are generally described in Draghici, 2003, Data AnalysisTools for DNA Microarrays, Chapman & Hall/CRC, which is herebyincorporated by reference in its entirety.

RT-PCR

In certain embodiments, to determine the feature values of biomarkers ina biomarker profile of level of expression of one or more of the markersdescribed herein, the feature values are measured by amplifying RNA froma sample using reverse transcription (RT) in combination with thepolymerase chain reaction (PCR). In accordance with this embodiment, thereverse transcription may be quantitative or semi-quantitative. TheRT-PCR methods taught herein may be used in conjunction with themicroarray methods described above. For example, a bulk PCR reaction maybe performed, and the PCR products may be resolved and used as probespots on a microarray.

Total RNA, or mRNA is used as a template and a primer specific to thetranscribed portion of the marker(s) is used to initiate reversetranscription. Methods of reverse transcribing RNA into cDNA are wellknown and described in Sambrook et al., 2001, supra. Primer design canbe accomplished based on known nucleotide sequences that have beenpublished or available from any publicly available sequence databasesuch as GenBank. For example, primers may be designed for any of themarkers described herein. Further, primer design may be accomplished byutilizing commercially available software (e.g., Primer Designer 1.0,Scientific Software etc.). The product of the reverse transcription issubsequently used as a template for PCR.

PCR provides a method for rapidly amplifying a particular nucleic acidsequence by using multiple cycles of DNA replication catalyzed by athermostable, DNA-dependent DNA polymerase to amplify the targetsequence of interest. PCR requires the presence of a nucleic acid to beamplified, two single-stranded oligonucleotide primers flanking thesequence to be amplified, a DNA polymerase, deoxyribonucleosidetriphosphates, a buffer and salts. The method of PCR is well known inthe art. PCR, is performed, for example, as described in Mullis andFaloona, 1987, Methods Enzymol. 155:335, which is hereby incorporated byreference in its entirety.

PCR can be performed using template DNA or cDNA (at least 10 fg; moreusefully, 1-1000 ng) and at least 25 pmol of oligonucleotide primers. Atypical reaction mixture includes: 2 μl of DNA, 25 pmol ofoligonucleotide primer, 2.5 μl of 10 M PCR buffer 1 (Perkin-Elmer,Foster City, Calif.), 0.4 μl of 1.25 M dNTP, 0.15 μl (or 2.5 units) ofTaq DNA polymerase (Perkin Elmer, Foster City, Calif.) and deionizedwater to a total volume of 25 μl. Mineral oil is overlaid and the PCR isperformed using a programmable thermal cycler.

Quantitative RT-PCR (“QRT-PCR”), which is quantitative in nature, canalso be performed to provide a quantitative measure of marker expressionlevels. In QRT-PCR reverse transcription arid PCR can be performed intwo steps, or reverse transcription combined with PCR can be performedconcurrently. One of these techniques, for which there are commerciallyavailable kits such as Taqman (Perkin Elmer, Foster City, Calif.) or asprovided by Applied Biosystems (Foster City, Calif.) is performed with atranscript-specific antisense probe. This probe is specific for the PCRproduct (e.g. a nucleic acid fragment derived from a gene) and isprepared with a quencher and fluorescent reporter probe complexed to the5′ end of the oligonucleotide. Different fluorescent markers areattached to different reporters, allowing for measurement of twoproducts in one reaction. When Taq DNA polymerase is activated, itcleaves off the fluorescent reporters of the probe bound to the templateby virtue of its 5′-to-3′ exonuclease activity. In the absence of thequenchers, the reporters now fluoresce. The color change in thereporters is proportional to the amount of each specific product and ismeasured by a fluorometer; therefore, the amount of each color ismeasured and the PCR product is quantified. The PCR reactions areperformed in 96-well plates so that samples derived from manyindividuals are processed and measured simultaneously. The Taqman systemhas the additional advantage of not requiring gel electrophoresis andallows for quantification when used with a standard curve.

A second technique useful for detecting PCR products quantitatively isto use an intercalating dye such as the commercially availableQuantiTect SYBR Green PCR (Qiagen, Valencia Calif.). RT-PCR is performedusing SYBR green as a fluorescent label which is incorporated into thePCR product during the PCR stage and produces a fluorescenceproportional to the amount of PCR product.

Both Taqman and QuantiTect SYBR systems can be used subsequent toreverse transcription of RNA. Reverse transcription can either beperformed in the same reaction mixture as the PCR step (one-stepprotocol) or reverse transcription can be performed first prior toamplification utilizing PCR (two-step protocol). Additionally, othersystems to quantitatively measure mRNA expression products are known,including Molecular Beacons®, which uses a probe having a fluorescentmolecule and a quencher molecule, the probe capable of forming a hairpinstructure such that when in the hairpin form, the fluorescence moleculeis quenched, and when hybridized the fluorescence increases giving aquantitative measurement of gene expression.

Northern Blot Assays

Any hybridization technique known to those of skill in the art can beused to generate feature values for biomarkers a biomarker profile. Inother particular embodiments, feature values for biomarkers in abiomarker profile can be obtained by Northern blot analysis (to detectand quantify specific RNA molecules. A standard Northern blot assay canbe used to ascertain an RNA transcript size, identify alternativelyspliced RNA transcripts, and the relative amounts of one or more genesdescribed herein (in particular, mRNA) in a sample, in accordance withconventional Northern hybridization techniques known to those persons ofordinary skill in the art. In Northern blots, RNA samples are firstseparated by size via electrophoresis in art agarose gel underdenaturing conditions. The RNA is then transferred to a membrane,cross-linked and hybridized with a labeled probe. Non-isotopic or highspecific activity radiolabeled probes can be used includingrandom-primed, nick-translated, or PCR-generated DNA probes, in vitrotranscribed RNA probes, and oligonucleotides. Additionally, sequenceswith only partial homology (e.g., cDNA from a different species orgenomic DNA fragments that might contain an exon) may be used as probes.The labeled probe, e.g., a radiolabelled cDNA, either containing thefull-length, single stranded DNA or a fragment of that DNA sequence maybe at least 20, at least 30, at least 50, or at least 100 consecutivenucleotides in length. The probe can be labeled by any of the manydifferent methods known to those skilled in this art. The labels mostcommonly employed for these studies are radioactive elements, enzymes,chemicals that fluoresce when exposed to ultraviolet light, and others.A number of fluorescent materials are known and can be utilized aslabels. These include, but are not limited to, fluorescein, rhodamine,auramine, Texas Red, AMCA blue and Lucifer Yellow. The radioactive labelcan be detected by any of the currently available counting procedures.Non-limiting examples of isotopes include ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr,⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re. Enzyme labels are likewiseuseful, and can be detected by any of the presently utilizedcolorimetric, spectrophotometric, fluorospectrephotometric, amperometricor gasometric techniques. The enzyme is conjugated to the selectedparticle by reaction with bridging molecules such as carbodiimides,diisocyanates, glutaraldehyde and the like. Any enzymes known to one ofskill in the art can be utilized. Examples of such enzymes include, butare not limited to, peroxidase, beta-D-galactosidase, urease, glucoseoxidase plus peroxidase and alkaline phosphatase, U.S. Pat. Nos.3,654,090, 3,850,752, and 4,016,043 are referred to by way of examplefor their disclosure of alternate labeling material and methods.

Methods of Detecting Proteins

In specific embodiments of the invention, feature values of biomarkersin a biomarker profile can be obtained by detecting proteins, forexample, by detecting the expression product (e.g., a nucleic acid orprotein) of one or more markers described herein, orpost-translationally modified, or otherwise modified, or processed formsof such proteins. In a specific embodiment, a biomarker profile isgenerated by detecting and/or analyzing one or more proteins and/ordiscriminating fragments thereof expressed from a marker disclosedherein using any method known to those skilled in the art for detectingproteins including, but not limited to protein microarray analysis,immunohistochemistry and mass spectrometry.

Standard techniques may be utilized for determining the amount of theprotein or proteins of interest present in a cell culture. For example,standard techniques can be employed using, e.g., immunoassays such as,for example, Western blot, immunoprecipitation followed by sodiumdodecyl sulfate polyacrylamide gel electrophoresis, (SDS-PAGE),immunocytochemistry, and the like to determine the amount of protein orproteins of interest present in a sample. One exemplary agent fordetecting a protein of interest is an antibody capable of specificallybinding to a protein of interest, preferably an antibody detectablylabeled, either directly or indirectly.

For such detection methods, if desired a protein from the cell cultureto be analyzed can easily be isolated using techniques which are wellknown to those of skill in the art. Protein isolation methods can, forexample, be such as those described in Harlow and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y.), which is hereby incorporated by reference inits entirety.

In certain embodiments, methods of detection of the protein or proteinsof interest involve their detection via interaction with aprotein-specific antibody. For example, antibodies directed to a proteinof interest. Antibodies can be generated utilizing standard techniqueswell known to those of skill in the art. In specific embodiments,antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or an antibody fragment (e.g., scFv, Fab or F(ab′)₂) can, forexample, be used.

For example, antibodies, or fragments of antibodies, specific for aprotein of interest can be used to quantitatively or qualitativelydetect the presence of a protein. This can be accomplished, for example,by immunofluorescence techniques. Antibodies (or fragments thereof) can,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of a protein ofinterest. In situ detection can be accomplished by removing a biologicalsample (e.g., a biopsy specimen) from a patient, and applying thereto alabeled antibody that is directed to a protein of interest. The antibody(or fragment) is preferably applied by overlaying the antibody (orfragment) onto a biological sample. Through the use of such a procedure,it is possible to determine not only the presence of the protein ofinterest, but also its distribution, in a particular sample. A widevariety of well-known histological methods (such as staining procedures)can be utilized to achieve such in situ detection.

Immunoassays for a protein of interest typically comprise incubating asample of a detectably labeled antibody capable of identifying a proteinof interest, and detecting the bound antibody by any of a number oftechniques well-known in the art. As discussed in more detail, below,the term “labeled” can refer to direct labeling of the antibody via,e.g., coupling (i.e., physically linking) a detectable substance to theantibody, and can also refer to indirect labeling of the antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody.

The sample can be brought in contact with and immobilized onto a solidphase support or carrier such as nitrocellulose, or other solid supportwhich is capable of immobilizing cells, cell particles or solubleproteins. The support can then be washed with suitable buffers followedby treatment with the detectably labeled fingerprint gene-specificantibody. The solid phase support can then be washed with the buffer asecond time to remove unbound antibody. The amount of bound label onsolid support can then be detected by conventional methods.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One of the ways in which an antibody specific for a protein of interestcan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, 1978, “The Enzyme Linkedimmunosorbent Assay (ELISA)”, Diagnostic Horizons 2:1-7, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.; Voller et al.,1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol.73:482-523; Maggio (ed.), 1980, Enzyme Immunoassay, CRC Press, BocaRaton, Fla.; Ishikawa et al., (eds.), 1981, Enzyme Immunoassay, KgakuShoin, Tokyo, each of which is hereby incorporated by reference in itsentirety). The enzyme which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric of by visual means. Enzymeswhich can be used to detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect a protein of interestthrough the use of a radioimmunoassay (RIA) (see, for example,Weintraub, 1986, Principles of Radioimmunoassays, Seventh TrainingCourse on Radioligand Assay Techniques, The Endocrine Society, which ishereby incorporated by reference in its entirety). The radioactiveisotope (e.g., ¹²⁵I, ¹³¹I, ³⁵S or ³H) can be detected by such means asthe use of a gamma counter or a scintillation counter or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetreaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction, Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescentfound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

In another embodiment, specific binding molecules other than antibodies,such as aptamers, may be used to bind the biomarkers. In yet anotherembodiment, the biomarker profile may comprise a measurable aspect of aninfectious agent (e.g., lipopolysaccharides or viral proteins) or acomponent thereof.

In some embodiments, a protein chip assay (e.g., The ProteinChip®Biomarker System, Ciphergen, Fremont, Calif.) is used to measure featurevalues for the biomarkers in the biomarker profile. See also, forexample, Lin, 2004, Modern Pathology, 1-9; Li, 2004, Journal of Urology171, 1782-1767; Wadsworth, 2004, Clinical Cancer Research, 10,1625-1632; Prieto, 2003, Journal of Liquid Chromatography & RelatedTechnologies 26, 2315-2328; Coombes, 2003, Clinical Chemistry 49,1615-1623; Mian, 2003, Proteomics 3, 1725-1737; Lehre et al., 2003, BJUInternational 92, 223-225; and Diamond, 2003, Journal of the AmericanSociety for Mass Spectrometry 14, 760-765, each of which is herebyincorporated by reference in its entirety.

In some embodiments, a bead assay is used to measure feature values forthe biomarkers in the biomarker profile. One such bead assay is theBecton Dickinson Cytometric Bead Array (CBA), CBA employs a series ofparticles with discrete fluorescence intensities to simultaneouslydetect multiple soluble analytes. CBA is combined with flow cytometry tocreate a multiplexed assay. The Becton Dickinson CBA system, as embodiedfor example in the Becton Dickinson Human Inflammation Kit, uses thesensitivity of amplified fluorescence detection by flow cytometrymeasure soluble analytes in a particle-based immunoassay. Each bead in aCBA provides a capture surface for a specific protein and is analogousto an individually coated well in an ELISA plate. The BD CBA capturebead mixture is in suspension to allow for the detection of multipleanalytes in a small volume sample.

In some embodiments, the multiplex analysis method described in U.S.Pat. No. 5,981,180 (“the '180 patent”), hereby incorporated by referencein its entirety, and in particular for its teachings of the generalmethodology, bead technology, system hardware and antibody detection, isused to measure feature values for the biomarkers in a biomarkerprofile. For this analysis, a matrix of microparticles is synthesized,where the matrix consists of different sets of microparticles. Each setof microparticles can have thousands of molecules of a distinct antibodycapture reagent immobilized on the microparticle surface and can becolor-coded by incorporation of varying amounts of two fluorescent dyes.The ratio of the two fluorescent dyes provides a distinct emissionspectrum for each set of microparticles, allowing the identification ofa microparticle set following the pooling of the various sets ofmicroparticles. U.S. Pat. Nos. 8,268,222 and 6,599,331 also are herebyincorporated by reference in their entirety, and in particular for theirteachings of various methods of labeling microparticles for multiplexanalysis.

Use of Other Methods of Detection

In some embodiments, a separation method may be used to determinefeature values for biomarkers in a biomarker profile, such that only asubset of biomarkers within the sample is analyzed. For example, thebiomarkers that are analyzed in a sample may be mRNA species from acellular extract which has been fractionated to obtain only the nucleicacid biomarkers within the sample, or the biomarkers may be from afraction of the total complement of proteins within the sample, whichhave been fractionated by chromatographic techniques.

Feature values for biomarkers in a biomarker profile can also, forexample, be generated by the use of one or more of the following methodsdescribed below. For example, methods may include nuclear magneticresonance (NMR) spectroscopy, a mass spectrometry method, such aselectrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS,ESI-MS/MS)^(n) (n is an integer greater than zero), matrix-assistedlaser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS), surface-enhanced laser desorption/ionizationtime-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionizationon silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupoletime-of-flight (Q-TOF), atmospheric pressure chemical ionization massspectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)^(n), atmospheric pressurephotoionization mass spectrometry (APPI-MS), APPI-MS/MS, andAPPI-(MS)^(n). Other mass spectrometry methods may include, inter glia,quadrupole, Fourier transform mass spectrometry (FTMS) and ion trap.Other suitable methods may include chemical extraction partitioning,column chromatography, ion exchange chromatography, hydrophobic (reversephase) liquid chromatography, isoelectric focusing, one-dimensionalpolyacrylamide gel electrophoresis (PAGE), two-dimensionalpolyacrylamide gel electrophoresis (2D-PAGE) or other chromatography,such as thin-layer, gas or liquid chromatography, or any combinationthereof. In one embodiment, the biological sample may be fractionatedprior to application of the separation method.

In one embodiment, laser desorption/ionization time-of-flight massspectrometry is used to determine feature values in a biomarker profilewhere the biomarkers are proteins or protein fragments that have beenionized and vaporized off an immobilizing support by incident laserradiation and the feature values are the presence or absence of peaksrepresenting these fragments in the mass spectra profile. A variety oflaser desorption/ionization techniques are known in the art (see, e.g.,Guttman et al., 2001, Anal. Chem. 73:1252-62 and Wei et al., 1999,Nature 399:243-246, each of which is hereby incorporated by reference inits entirety).

Laser desorption/ionization time-of-flight mass spectrometry allows thegeneration of large amounts of information in a relatively short periodof time. A biological sample is applied to one of several varieties of asupport that binds all of the biomarkers, or a subset thereof, in thesample. Cell lysates or samples are directly applied to these surfacesin volumes as small as 0.5 μL, with or without prior purification orfractionation. The lysates or sample can be concentrated or dilutedprior to application onto the support surface. Laserdesorption/ionization is then used to generate mass spectra of thesample, or samples, in as little as three hours.

Data Analysis Algorithms

Biomarker expression profile of T-MSC are factors discriminating betweenclinical grade T-MSC and non-clinical grade T-MSC. The identity of thesebiomarkers and their corresponding features (e.g., expression levels)can be used to develop a decision rule, or plurality of decision rules,that discriminate between clinical grade and non-clinical grade T-MSC.Specific data analysis algorithms for building a decision rule, orplurality of decision rules, can discriminate between clinical gradeT-MSC and non-clinical grade T-MSC. Once a decision rule has been builtusing these exemplary data analysis algorithms or other techniques knownin the art, the decision rule can be used to classify a T-MSC populationinto one of the two or more phenotypic classes (e.g., a clinical gradeor a non-clinical grade T-MSC). This is accomplished by applying thedecision rule to a biomarker profile obtained from the cell culture.Such decision rules, therefore, have enormous value as defining thequality of T-MSC.

In a certain embodiment, provided herein is a method for the evaluationof a biomarker profile from a test cell culture compared to biomarkerprofiles obtained from a cell culture in a control population. In someembodiments, each biomarker profile obtained from the controlpopulation, as well as the test cell culture, comprises a feature foreach of a plurality of different biomarkers. In some embodiments, thiscomparison is accomplished by (i) developing a decision rule using thebiomarker profiles from the control population and (ii) applying thedecision rule to the biomarker profile from the test cell culture. Assuch, the decision rules applied in some embodiments of the presentinvention are used to determine whether a test cell culture is clinicalgrade or non-clinical grade. In certain embodiments, the controlpopulation is a clinical grade T-MSC. In other embodiments, the controlpopulation is BM-MSC.

In some embodiments of the present invention, when the results of theapplication of a decision rule indicate that the test cell culture isclinical grade T-MSC, it is used for treatment. If the results of anapplication of a decision rule indicate that the test cell culture isnon-clinical grade T-MSC, the test cell culture is not used fortreatment.

Modification of T-MSC

Provided herein is a method of modifying mesenchymal stem cells toproduce a population of modified MSC that has improved immunosuppressivefunction. The MSC have the following characteristics: (i) contain >95%of cells expressing group-1 markers; (ii) contain >80% of cellsexpressing group 2 markers; (iii) contain <5% of cells expressinggroup-3 markers; (iv) expresses IL-10 and TGFβ; (v) contain <2% of cellsexpressing IL-6, IL-12 and TNFα; and (vi) contains <0.001% of cellsco-expressing all group-4 markers, wherein group-1 markers are CD73,CD90, CD105, CD146, CD166, and CD44, group-2 markers are CD13, CD29,CD54, CD49E, group-3 markers are CD45, CD34, CD31 and SSEA4, and group-4markers are OCT4, NANOG, TRA-1-60 and SSEA4.

Provided herein is a method of increasing immunosuppressive function ofT-MSC try increasing the expression of AIF. In an embodiment, the methodcomprises decreasing the expression of PIF. In an embodiment, the methodcomprises decreasing the expression of IL6, IL12, TNFα, RAGE and otherPIF in T-MSC. In an embodiment, the method comprises increasing theexpression of TGFβ and IL-10 in T-MSC.

In certain embodiments, the method comprises genetic and epigeneticmodifications of T-MSC that are known in the art. In certainembodiments, the genetic modification or epigenetic regulation includes,but is not limited to, knockout, small hair pin RNA (“shRNA”), micro RNA(“miRNA”), non-coding RNA (“ncRNA”), mopholino oligo, decoy RNA, DNAmethylation regulation, histone methylation regulation, translationinhibition and/or antibody blocking. In certain embodiments, MSC aremodified through transposomes, toll-like receptor ligands, or smallmolecules.

In certain embodiments, small molecules are used to target any of thesignaling pathway components of IL-6 signaling. In certain embodiments,the target includes, but is not limited to, gp130, STAT3, Cathepsin S,NFkappaB, IRF5. In certain embodiments, IL-12 expression is decreased inT-MSC by activation of the prostaglandin E2 pathway, by increasingintracellular cyclic AMP levels with cAMP agonists that include, but arenot limited to, forskolin, cholera toxin, β1- and β2 adrenoreceptoragonists, by inhibition of the NF-κB Rel-B pathway, by treating T-MSCwith apoptotic cells, by treatment with phosphatidylserine, by treatmentwith butyrate, by treatment with Triptolide or extracts fromTripterygium wilfordii or synthetic forms or Triptolide (i.e.,Minnelide).

In certain embodiments, MSC may be modified to express a certain markerusing methods known in the art of recombinant DNA. In certainembodiments, MSC may be modified by transfection using the nucleotidesequence encoding the marker. The marker can be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. The necessary transcriptional and translationalelements can also be present. The regulatory regions and enhancerelements can be of a variety of origins, both natural and synthetic. Avariety of host-vector systems may be utilized to express the marker.These include, but are not limited to, mammalian cell systems infectedwith virus (e.g., vaccinia virus, adenovinis, etc.); insect cell systemsinfected with virus (e.g., baculovirus); microorganisms such as yeastcontaining yeast vectors, or bacteria transformed with bacteriophage,DNA, plasmid DNA, or cosmid DNA; and stable cell lines generated bytransformation using a selectable marker. The expression elements ofvectors vary in their strengths and specificities. Depending on thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used.

Once a vector encoding the appropriate marker has been synthesized, theMSC is transformed or transfected with the vector of interest.

Standard methods of introducing a nucleic add sequence of interest intothe MSC can be used. Transformation may be by any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus and transducing a host cell withthe virus, and by direct uptake of the polynucleotide. Mammaliantransformations (i.e., transfections) by direct uptake may be conductedusing the calcium phosphate precipitation method of Graham & Van der Eb.1978, Virol. 52:546, or the various known modifications thereof. Othermethods for introducing recombinant polynucleotides into cells,particularly into mammalian cells, include dextran-mediatedtransfection, calcium phosphate mediated transfection, polybrenemediated transfection, protoplast fusion, electroporation, encapsulationof the polynucleotide(s) in liposomes, and direct microinjection of thepolynucleotides into nuclei. Such methods are well-known to one of skillin the art.

In a preferred embodiment, stable cell lines containing the constructsof interest are generated for high throughput screening. Such stablecells lines may be generated by introducing a construct comprising aselectable marker, allowing the cells to grow for 1-2 days in anenriched medium, and then growing the cells on a selective medium. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); got, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30:147) genes.

Stem Cell Collection Composition

The stem cell collection composition can comprise anyphysiologically-acceptable solution suitable for the collection and/orculture of stem cells, for example, a saline solution (e.g.,phosphate-buffered saline, Kreb's solution, modified Kreb's solution,Eagle's solution, 0.9% NaCl, etc.), a culture medium (e.g., DMEM,H.DMEM, etc.), and the like.

The stem cell collection composition can comprise one or more componentsthat tend to preserve stem cells, that is, prevent the stem cells fromdying, or delay the death of the stem cells, reduce the number of stemcells in a population of cells that die, or the like, from the time ofcollection to the time of culturing. Such components can be, e.g., anapoptosis inhibitor (e.g., a caspase inhibitor or JNK inhibitor); avasodilator (e.g., magnesium sulfate, an antihypertensive drug, atrialnatriuretic peptide (ANP), adrenocorticotropin, corticotropin-releasinghormone, sodium nitroprusside, hydralazine, adenosine triphosphate,adenosine, indomethacin or magnesium sulfate, a phosphodiesteraseinhibitor, etc.); a necrosis inhibitor (e.g.,2-(1H-Indol-3-yl)-3-pentylamino-maleimide, pyrrolidine dithiocarbamate,or clonazepam); a TNF-α inhibitor; and/or an oxygen-carryingperfluorocarbon perfluorooctyl bromide, perfluorodecyl bromide, etc.).

The stem cell collection composition can comprise one or moretissue-degrading enzymes, e.g., a metalloprotease, a serine protease, aneutral protease, an RNase, or a DNase, or the like. Such enzymesinclude, but are not limited to, collagenases (e.g., collagenase I, II,III or IV, a collagenase from Clostridium histolyticum, etc.); dispose,thermolysin, elastase, trypsin, LIBERASE, hyaluronidase, and the like.

The stem cell collection composition can comprise a bacteriocidally orbacteriostatically effective amount of an antibiotic. In certainnon-limiting embodiments, the antibiotic is a macrolide (e.g.,tobramycin), a cephalosporin (e.g., cephalexin, cephradine, cefuroxime,cefprozil, cefaclor, cefixime or cefadroxil), a clarithromycin, anerythromycin, a penicillin (e.g., penicillin V) or a quinolone (e.g.,ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, astreptomycin, etc. In a particular embodiment, the antibiotic is activeagainst Gram(+) and/or Gram(−) bacteria, e.g., Pseudomonas aeruginosa,Staphylococcus aureus, and the like.

The stem cell collection composition can also comprise one or more ofthe following compounds: adenosine. (about 1 mM to about 50 mM);D-glucose (about 20 mM to about 100 mM); magnesium ions (about 1 mM toabout 50 mM); a macromolecule of molecular weight greater than 20,000daltons, in one embodiment, present in an amount sufficient to maintainendothelial integrity and cellular viability (e.g., a synthetic ornaturally occurring colloid, a polysaccharide such as dextran or apolyethylene glycol present at about 25 g/l to about 100 g/l, or about40 g/l to about 60 g/l); an antioxidant (e.g., butylated hydroxyanisole,butylated hydroxytoluene, glutathione, vitamin C or vitamin E present atabout 25 μM to about 100 μM); a reducing agent (e.g., N-acetylcysteinepresent at about 0.1 mM to about 5 mM); an agent that prevents calciumentry into cells (e.g., verapamil present at about 2 μM to about 25 μM);nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant,in one embodiment, present in an amount sufficient to help preventclotting of residual blood (e.g., heparin or hirudin present at aconcentration of about 1000 units/1 to about 100,000 units/l); or anamiloride containing compound (e.g., amiloride, ethyl isopropylamiloride, hexamethylene amiloride, dimethyl amiloride or isobutylamiloride present at about 1.0 μM to about 5 μM).

Immunomodulation Using T-MSC

Provided herein is the modulation of the activity (e.g. reduced cellproliferation, reduced cell survival, impaired cell migration to sitesof inflammation, reduced ability of the cells to promote or prolonginflammation or enhanced cell functions that promote the restoration ofhealthy tissue or organ homeostasis) of an immune cell, or plurality ofimmune cells, by contacting the immune cell(s) with a plurality of T-MSCor iT-MSC. In one embodiment, the method of modulating an immuneresponse comprises contacting a plurality of immune cells with aplurality of T-MSC or iT-MSC for a time sufficient for the T-MSC oriT-MSC to detectably suppress an immune response, wherein the T-MSC oriT-MSC detectably suppress T cell proliferation in a mixed lymphocytereaction (MLR) assay.

Since BM-MSC or other adult tissue derived MSC have been used to treatmany autoimmune diseases. BM-MSC are also used for tissue repairing bylimiting inflammation and secret growth arid protective factors, andreplacing damaged tissues. As shown later in the examples, T-MSC havesuperior immunosuppressive function to BM-MSC, and thus T-MSC can beused in all areas and diseases that are currency targeted by BM-MSC.

T-MSC or iPS-MSC used for immunomodulation may be derived or obtainedfrom an embryonic stem cell line or induced pluripotent stem cell line,respectively. T-MSC or iPS-MSC used for immunomodulation may also bederived from the same species as the immune cells whose activity is tobe modulated or from a different species as that of the immune cellswhose activity is to be modulated.

An “immune cell” in the context of this method means any cell of theimmune system, particularly T cells and NK (natural killer) cells. Thus,in various embodiments of the method, T-MSC are contacted with aplurality of immune cells., wherein the plurality of immune cells are,or comprise, a plurality of T cells (e.g., a plurality of CD3⁺ T cells,CD4⁺ T cells and/or CD8⁺ T cells) and/or natural killer cells. An“immune response” in the context of the method can be any response by animmune cell to a stimulus normally perceived by an immune cell, e.g., aresponse to the presence of an antigen. In various embodiments, animmune response can be the proliferation of T cells (e.g., CD3⁺ T cells,CD4⁺ T cells and/or CD8⁺ T cells) in response to a foreign antigen, suchas an antigen present in a transfusion or graft, or to a self-antigen,as in an autoimmune disease. The immune response can also be aproliferation of T cells contained within a graft. The immune responsecan also be any activity of a natural killer (NK) cell, the maturationof a dendritic cell, or the like. The immune response can also be alocal, tissue- or organ-specific, or systemic effect of an activity ofone or more classes of immune cells, e.g., the immune response can begraft versus host disease, inflammation, formation ofinflammation-related scar tissue, an autoimmune condition (e.g.,rheumatoid arthritis, Type I diabetes, lupus erythematosus, etc.), andthe like.

“Contacting” in this context encompasses bringing the T-MSC and immunecells together in a single container (e.g., culture dish, flask, vial,etc.) or in vivo, for example, the same individual (e.g., mammal, forexample, human). In a preferred embodiment, the contacting is for a timesufficient, and with a sufficient number of T-MSC and immune cells, thata change in an immune function of the immune cells is detectable. Morepreferably, in various embodiments, the contacting is sufficient tosuppress immune function (e.g., T cell proliferation in response to anantigen) by at least 50%, 60%, 70%, 80%, 90% or 95%, compared to theimmune function in the absence of the T-MSC. Such suppression in an inviva context can be determined in an in vitro assay; that is, the degreeof suppression in the in vitro assay can be extrapolated, for aparticular number of T-MSC and a number of immune cells in a recipientindividual, to a degree of suppression in the individual.

The invention in certain embodiments provides methods of using T-MSC tomodulate an immune response, or the activity of a plurality of one ormore types of immune cells, in vitro. Contacting the T-MSC and pluralityof immune cells can comprise combining the T-MSC and immune cells in thesame physical space such that at least a portion of the plurality ofT-MSC interacts with at least a portion of the plurality of immunecells; maintaining the T-MSC and immune cells in separate physicalspaces with common medium or can comprise contacting medium from one ora culture of T-MSC or immune cells with the other type of cell (forexample, obtaining culture medium from a culture of T-MSC andresuspending isolated immune cells in the medium). In a specificexample, the contacting is a Mixed Lymphocyte Reaction (MLR),

Such contacting can, for example, take place in an experimental settingdesigned to determine the extent to which a particular plurality ofT-MSC is immunomodulatory, e.g., immunosuppressive. Such an experimentalsetting can be, for example, a mixed lymphocyte reaction (MLR) orregression assay. Procedures for performing the MLR and regressionassays are well-known in the art. See, e.g., Schwarz, “The MixedLymphocyte Reaction: An in Vitro Test for Tolerance,” J. Exp. Med.127(5):879-890 (1965); Lacerda et al., “Human Epstein-Barr Virus(EBV)-Specific Cytotoxic T Lymphocytes Home Preferentially to and InduceSelective Regressions of Autologous EBV-Induced B Lymphoproliferationsin Xenografted C.B-17 Scid/Scid Mice,” J. Exp. Med. 183:1215-1228(1996). In a preferred embodiment, an MLR is performed in which aplurality of T-MSC are contacted with a plurality of immune cells (e.g.,lymphocytes, for example, CD3⁺ CD4⁺ and/or CD8⁺ T lymphocytes).

The MLR can be used to determine the immunosuppressive capacity of aplurality of T-MSC. For example, a plurality of T-MSC can be tested inan MLR comprising combining CD4⁺ or CD8⁺ cells, dendritic cells (DC) andT-MSC in a ratio of about 10:1:2, wherein the T cells are stained with adye such as, e.g., CFSE that partitions into daughter cells, and whereinthe T cells are allowed to proliferate for about 6 days. The pluralityof T-MSC is immunosuppressive if the T cell proliferation at 6 days inthe presence of T-MSC is detectably reduced compared to T cellproliferation in the presence of DC and absence of T-MSC. In such anMLR, T-MSC are either thawed or harvested from culture. About 10,000T-MSC are resuspended in 100 μl of medium (RPMI, 1640, 1 mM HEPESbuffer, antibiotics, and 5% pooled human serum), and allowed to attachto the bottom of a well for 2 hours. CD4⁺ and/or CD8⁺ T cells areisolated from whole peripheral blood mononuclear cells with Miltenyimagnetic beads. The cells are CFSE stained, and a total of 100,000 Tcells (CD4⁺ cells alone, CD8⁺ T cells alone, or equal amounts of CD4⁺and CD8⁺ T cells) are added per well. The volume in the well is broughtto 200 μl, and the MLR is allowed to proceed.

In one embodiment, therefore, the invention provides a method ofsuppressing an immune response comprising contacting a plurality ofimmune cells with a plurality of T-MSC for a time sufficient for theT-MSC to detectably suppress T cell proliferation in a mixed lymphocytereaction (MLR) assay.

Populations of T-MSC obtained from different embryonic stem cell lines,can differ in their ability to modulate an activity of an immune cell,e.g., can differ in their ability to suppress T cell activity orproliferation or NK cell activity. It is thus desirable to determine,prior to use, the capacity of a particular population of T-MSC forimmunosuppression. Such a capacity can be determined, for example, bytesting a sample of the stem cell population in an MLR or regressionassay. In one embodiment, an MLR is performed with the sample, and adegree of immunosuppression in the assay attributable to the T-MSC isdetermined. This degree of immunosuppression can then be attributed tothe stem cell population that was sampled. Thus, the MLR can be used asa method of determining the absolute and relative ability of aparticular population of T-MSC to suppress immune function. Theparameters of the MLR can be varied to provide more data or to bestdetermine the capacity of a sample of T-MSC to immunosuppress. Forexample, because immunosuppression by T-MSC appears to increase roughlyin proportion to the number of T-MSC present in the assay, the MLR canbe performed with, in one embodiment, two or more numbers of stem cells,e.g., 1×10³, 3×10³, 1×10⁻¹ and/or 3×10⁻⁴ T-MSC per reaction. The numberof T-MSC relative to the number of T cells in the assay can also bevaried. For example, T-MSC and T cells in the assay can be present inany ratio of, e.g., about 10:1 to about 1:10, preferably about 1:5,though a relatively greater number of T-MSC or T cells can be used.

The invention also provides methods of using T-MSC to modulate an immuneresponse, or the activity of a plurality of one or more types of immunecells, in vivo. T-MSC and immune cells can be contacted, e.g., in anindividual that is a recipient of a plurality of T-MSC. Where thecontacting is performed in an individual, in one embodiment, thecontacting is between exogenous T-MSC (that is, T-MSC not derived fromthe individual) and a plurality of immune cells endogenous to theindividual, In specific embodiments, the immune cells within theindividual are CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and/or NKcells,

Such immunosuppression using T-MSC would be advantageous for anycondition caused or worsened by, or related to, an inappropriate orundesirable immune response. T-MSC-mediated immunomodulation, e.g.,immunosuppression, would, for example, be useful in the suppression ofan inappropriate immune response raised by the individual's immunesystem against one or more of its own tissues. In various embodiments,therefore, the invention provides a method of suppressing an immuneresponse, wherein the immune response is an autoimmune disease, e.g.,lupus erythematosus, diabetes, rheumatoid arthritis, or multiplesclerosis.

The contacting of the plurality of T-MSC with the plurality of one ormore types of immune cells can occur in vivo in the context of, or as anadjunct to, for example, grafting or transplanting of one or more typesof tissues to a recipient individual. Such tissues may be, for example,bone marrow or blood; an organ; a specific tissue (e.g., skin graft);composite tissue allograft (i.e., a graft comprising two or moredifferent types of tissues); etc. In this regard, the T-MSC can be usedto suppress one or more immune responses of one or more immune cellscontained within the recipient individual, within the transplantedtissue or graft, or both. The contacting can occur before, during and/orafter the grafting or transplanting. For example, T-MSC can beadministered at the time of the transplant or graft. The T-MSC can also,or alternatively, be administered prior to the transplanting orgrafting, e.g., about 1, 2, 3, 4, 5, 6 or 7 days prior to thetransplanting or grafting. T-MSC can also, or alternatively, beadministered to a transplant or graft recipient after thetransplantation or grafting, for example, about 1, 2, 3, 4, 5, 6 or 7days after the transplanting or grafting. Preferably, the plurality of Tcells are contacted with the plurality T-MSC before any detectable signor symptom of an immune response, either by the recipient individual orthe transplanted tissue or graft, e.g., a detectable sign or symptom ofgraft-versus-host disease or detectable inflammation, is detectable.

In another embodiment, the contacting within an individual is primarilybetween exogenous T-MSC and exogenous progenitor cells or stem cells,e.g., exogenous progenitor cells or stem cells that differentiate intoimmune cells. For example, individuals undergoing partial or fullimmunoablation myeloablation as an adjunct to cancer therapy can receiveT-MSC in combination with one or more other types of stem or progenitorcells. For example, the T-MSC can be combined with a plurality of CD34⁺cells, e.g., CD34⁺ hematopoietic stem cells. Such CD34⁺ cells can be,e.g., CD34⁺ cells from a tissue source such as peripheral blood,umbilical cord blood, placental blood, or bone marrow. The CD34⁺ cellscan be isolated from such tissue sources, or the whole tissue source(e.g., units of umbilical cord blood or bone marrow) or a partiallypurified preparation from the tissue source (e.g., white blood cellsfrom cord blood) can be combined with the T-MSC.

The T-MSC are administered to the individual preferably in a ratio, withrespect to the known or expected number of immune cells, e.g., T cells,in the individual, of from about 10:1 to about 1:10, preferably about1:5. However, a plurality of T-MSC can be administered to an individualin a ratio of in non-limiting examples, about 10,000:1, about 1,000:1,about 100:1, about 10:1, about 1:1, about 1:10, about 1:100, about1:1,000 or about 1:10,000. Generally, about 1×10⁵ to about 1×10⁸ T-MSCper recipient kilogram, preferably about 1×10⁵ to about 1×10⁷ T-MSCrecipient kilogram can be administered to effect immunosuppression. Invarious embodiments, a plurality of T-MSC administered to an individualor subject comprises at least, about, or no more than, 1×10⁵, 3×10⁵,1×10⁵, 3×10⁶, 1×10⁷, 3×10⁷, 1×10⁸, 3×10⁸, 1×10⁹, 3×10⁹ T-MSC, or more.

The T-MSC can also be administered with one or more second types of stemcells, e.g., mesenchymal stem cells from bone marrow. Such second stemcells can be administered to an individual with T-MSC in a ratio of,e.g., about 1:10 to about 10:1.

To facilitate contacting the T-MSC and immune cells in vivo, the T-MSCcan be administered to the individual by any route sufficient to bringthe T-MSC and immune cells into contact with each other. For example,the T-MSC can be administered to the individual, e.g., intravenously,intramuscularly, intraperitoneally, or directly into an organ, e.g.,pancreas For in vivo administration, the T-MSC can be formulated as apharmaceutical composition.

The method of immunosuppression can additionally comprise the additionof one or more immunosuppressive agents, particularly in the in vivacontext. In one embodiment, the plurality of T-MSC are contacted withthe plurality of immune cells in vivo in an individual, and acomposition comprising an immunosuppressive agent is administered to theindividual. Immunosuppressive agents are well known in the art andinclude, e.g., anti-T cell receptor antibodies (monoclonal orpolyclonal, or antibody fragments or derivatives thereof), anti-IL-2receptor antibodies (e.g., Basiliximab (SIMULECT®) or daclizumab(ZENAPAX®), anti T cell receptor antibodies (e.g., Muromonab-CD3),azathioprine, corticosteroids, cyclosporine, tacrolimus, mycophenolatemofetil, sirolimus, calcineurin inhibitors, and the like. In a specificembodiment, the immunosuppressive agent is a neutralizing antibody tomacrophage inflammatory protein (MIP)-1α or MIP-1β.

Preservation of T-MSC and/or T-MSC-DL

T-MSC and/or T-MSC-DL can be preserved, that is, placed under conditionsthat allow for long-term storage, or conditions that inhibit cell deathby, e.g., apoptosis or necrosis. T-MSC and/or T-MSC-DL can be preservedusing, e.g., a composition comprising an apoptosis inhibitor, necrosisinhibitor. In one embodiment, the invention provides a method ofpreserving a population of stem cells comprising contacting a populationof stem cells with a stem cell collection composition comprising aninhibitor of apoptosis, wherein the inhibitor of apoptosis is present inan amount and for a time sufficient to reduce or prevent apoptosis inthe population of stem cells, as compared to a population of stem cellsnot contacted with the inhibitor of apoptosis. In a specific embodiment,the inhibitor of apoptosis is a caspase inhibitor. In another specificembodiment, the inhibitor of apoptosis is a JNK inhibitor. In a morespecific embodiment, the JNK inhibitor does not modulate differentiationor proliferation of the stem cells. In another embodiment, the stem cellcollection composition comprises an inhibitor of apoptosis and anoxygen-carrying perfluorocarbon in separate phases. In anotherembodiment, the stem cell collection composition comprises an inhibitorof apoptosis and an oxygen-carrying perfluorocarbon in an emulsion. Inanother embodiment, the stem cell collection composition additionallycomprises an emulsifier, e.g., lecithin. In another embodiment, theapoptosis inhibitor and the perfluorocarbon are between about 0° C. andabout 25° C. at the time of contacting the stem cells. In another morespecific embodiment, the apoptosis inhibitor and the perfluorocarbon arebetween about 2° C. and 10° C., or between about 2° C. and about 5° C.,at the time of contacting the stem cells. In another more specificembodiment, the contacting is performed during transport of thepopulation of stem cells, In another more specific embodiment, thecontacting is performed during freezing and thawing of the population ofstem cells.

In another embodiment, the invention provides a method of preserving apopulation of T-MSC and/or T-MSC-DL comprising contacting the populationof stem cells with an inhibitor of apoptosis and an organ preservingcompound, wherein the inhibitor of apoptosis is present in an amount andfor a time sufficient to reduce or prevent apoptosis in the populationof stem cells, as compared to a population of stem cells not contactedwith the inhibitor of apoptosis.

Typically, during T-MSC and/or T-MSC-DL collection. enrichment andisolation, it is preferable to minimize or eliminate cell stress due tohypoxia and mechanical stress. In another embodiment of the method,therefore, a stem cell, or population of stem cells, is exposed to ahypoxic condition during collection, enrichment or isolation for lessthan six hours during the preservation, wherein a hypoxic condition is aconcentration of oxygen that is less than normal blood oxygenconcentration. In a more specific embodiment, the population of stemcells is exposed to the hypoxic condition. for less than two hoursduring the preservation. In another more specific embodiment, thepopulation of stem cells is exposed to the hypoxic condition for lessthan one hour, or less than thirty minutes, or is not exposed to ahypoxic condition, during collection, enrichment or isolation. Inanother specific embodiment, the population of stem cells is not exposedto shear stress during collection, enrichment or isolation.

The T-MSC and/or T-MSC-DL can be cryopreserved, e.g., incryopreservation medium in small containers, e.g., ampoules. Suitablecryopreservation medium includes, but is not limited to, culture mediumincluding, e.g., growth medium, or cell freezing medium, for examplecommercially available cell freezing medium, e.g., C2695, C2639 or C6039(Sigma). Cryopreservation medium preferably comprises DMSO(dimethylsulfoxide), at a concentration of, e.g., about 10% (v/v).Cryopreservation medium may comprise additional agents, for example,methylcellulose and/or glycerol. T-MSC and/or T-MSC-DL are preferablycooled at about 1° C./min during cryopreservation. A preferredcryopreservation temperature is about −80° C. to about −180° C.,preferably about −125° C. to about −140° C. Cryopreserved cells can betransferred to liquid nitrogen prior to thawing for use. In someembodiments, for example, once the ampoules have reached about −90° C.,they are transferred to a liquid nitrogen storage area. Cryopreservedcells preferably are thawed at a temperature of about 25° C. to about40° C., preferably to a temperature of about 37° C.

Cryopreserved T-MSC and/or T-MSC-DL

The T-MSC and/or T-MSC-DL disclosed herein can be preserved, forexample, cryopreserved for later use. Methods for cryopreservation ofcells, such as stem cells, are well known in the art. T-MSC and/orT-MSC-DL can be prepared in a form that is easily administrable to anindividual. For example, provided herein are T-MSC and/or T-MSC-DL thatare contained within a container that is suitable for medical use. Sucha container can be, for example, a sterile plastic bag, flask, jar, orother container from which the T-MSC and/or T-MSC-DL can be easilydispensed. For example, the container can be a blood bag or otherplastic, medically-acceptable bag suitable for the intravenousadministration of a liquid to a recipient. The container is preferablyone that allows for cryopreservation of the combined stem cellpopulation. Cryopreserved T-MSC and/or T-MSC-DL can comprise T-MSCand/or T-MSC-DL derived from a single donor, or from multiple donors.The T-MSC and/or T-MSC-DL can be completely HLA-matched to an intendedrecipient, or partially or completely HLA-mismatched.

In another specific embodiment, the container is a bag, flask, or jar.In a more specific embodiment, the bag is a sterile plastic bag. In amore specific embodiment, the bag is suitable for, allows or facilitatesintravenous administration of the T-MSC and/or T-MSC-DL. The bag cancomprise multiple lumens or compartments that are interconnected toallow mixing of the T-MSC and/or T-MSC-DL and one or more othersolutions, e.g., a drug, prior to, or during, administration. In anotherspecific embodiment, the composition comprises one or more compoundsthat facilitate cryopreservation of the combined stem cell population.In another specific embodiment, the T-MSC and/or T-MSC-DL is containedwithin a physiologically-acceptable aqueous solution. In a more specificembodiment, the physiologically-acceptable aqueous solution is a 0.9%NaCl solution. In another specific embodiment, the hES-MSC areHLA-matched to a recipient of the stem cell population. In anotherspecific embodiment, the combined stem cell population comprises hES-MSCthat are at least partially HLA-mismatched to a recipient of the stemcell population.

Differentiation of T-MSC into Multiple Lineages

T-MSC maybe differentiated into various cell lineages including neuronallineage cells or neurons, or adipocytes, or myoblasts, or fibroblasts,or osteoblasts or chrondrocytes. Unless specifically indicated, T-MSCmay be plated onto cell culture plates coated with gelatin, collagen,fibronectin, Matrigel, laminin, vitronectin, or poly(lysine). T-MSC maybe plated at a concentration of 1×10³ cells/cm² to 1×10⁴ cells/cm² inserum free medium or serum-containing medium with bovine serum FBS orABHS. T-MSCs plated according to the above mentioned conditions may bedifferentiated by one of the following methods.

In one embodiment, T-MSC may be differentiated in medium containing 1-50ng/mL Fibroblast Growth Factor (FGF)-2 (optimally 10 ng/ml) plus 1-50ng/ml Epidermal Growth Factor (EGF) (optimally 10 ng/ml) plus 0.5-5ng/ml Platelet-Derived Growth Factor (PDGF) (optimally 1 ng/ml). Themedium is changed every 2 to 3 days and the cells are harvested after2-4 weeks with an expected yield of 0.5×10⁶-2×10⁶ neuronal lineage cellsper 1×10⁶ T-MSC.

In another embodiment, T-MSC may be differentiated into neuronal lineagecells by plating on Poly-ornithine and Laminin coated plates. T-MSCswill be differentiated in three stages. Stage 1: 1-50 ng/ml FGF-2(optimally 10 ng/ml) and 1-50 ng/ml EGF (optimally 10 ng/ml), to primehMSCs towards a neural fate. Stage 2: 10-200 ng/ml Sonic Hedgehog (SHH)(optimally 100 ng/ml), 1-50 ng/ml FGF-8 (human) (optimally 10 ng/ml) and50-500 μM AAP (optimally 200 μM), for initiating midbrain specification.Stage 3: 5-500 ng/ml Glial-Derived Neurotrophic Factor (GDNF) (optimally50 ng/ml) and 50-500 μM AAP (optimally 200 μM), for inducingdifferentiation and maturation towards a dopaminergic neuronalphenotype. Each stage is applied for 1 week and the adherent cells arepassaged by disassociation with Trypsin or TrypLE/dispase between eachstage. Growth factors are replenished every day and the medium ischanged every 2 days. Expected yield is 0.5×10⁶-4×10⁶ neuronal lineagecells per 1×10⁶ T-MSC.

In another embodiment, T-MSC may be differentiated into neuronal lineagecells in Neurobasal medium (Gibco) containing 0.25×B-27 supplement plus10-200 ng/ml Sonic Hedgehog (SHH) (optimally 100 ng/ml), plus 1-50 ng/mlFGF-8 (mouse) (optimally 10 ng/ml) plus 1-200 ng/ml FGF-2 (optimally 50ng/ml). Cells are harvested after 6- and 12-days. Media is not replacedduring this period. Expected yield is 0.5×10⁶-4×10⁶ neuronal lineagecells per 1×10⁶ T-MSC.

In another embodiment, T-MSC may be differentiated into neuronal lineagecells in two stages. Stage 1: T-MSC are cultured in serum-free medium(DMEM) supplemented with 2 mM glutamine, 1-20 U/ml (optimally 12.5 U/ml)nystatin, N2 supplement, and 2-50 ng/ml (optimally 20 ng/ml) fibroblastgrowth factor-2 (FGF-2) and 1-50 ng/mL EGF (optimally 10 ng/ml) for48-72 hours. Stage 2: cells are cultured in Neurobasal medium plus B27supplement plus 0.1-10 mM (optimally 1 mM) dibutyryl cyclic AMP(dbcAMP), 3-isobutyl-1-methylxanthine (IBMX), and 10-500 μM (optimally200 μM) ascorbic acid plus 1-100 ng/ml BDNF (optimally 50 ng/ml), 1-50ng/ml glial-derived neurotrophic factor (GDNF; optimally 10 ng/ml),0.2-10 ng/ml transforming growth factor-β3 (TGF-β3, optimally 2 ng/ml),and 0.05-5 μM all-transretinoic acid (RA, optimally 0.1 μM). Each stageis applied for 1 week and the adherent cells are passaged bydisassociation with Trypsin or TrypLE/dispase between each stage. Themedium is changed every 2 days and the expected yield is 0.5×10⁶-4×10⁶neuronal lineage cells per 1×10⁶ T-MSC.

In another embodiment, T-MSC may be cultured to induce osteogenicdifferentiation. T-MSCs will be cultured in low glucose DMEM plus 10%FCS, 1-150 uM (optimally 80 μM) ascorbic acid 2-phosphate, 0.5-5 μM(optimally 1 μM) dexamethasone, and 1-100 mM (optimally 20 mM)beta-glycerophosphate. The medium is changed every 2 to 3 days and theexpected yield is 0.5×10⁶-4×10⁶ neuronal lineage cells per 1×10⁶ T-MSCafter 2 weeks.

In another embodiment, T-MSC may be cultured to induce adipogenicdifferentiation. T-MSCs will be grown in low glucose DMEM plus 20% FCS,1-10 μg/ml (optimally 5 μg/ml) insulin, 0.5-10 μM (optimally 2 μM)dexamethasone, 0.1-1 mM (optimally 0.5 mM) isobutylmethylxanthine, and1-100 μM (optimally 60 μM) indomethacin. The medium is changed every 2to 3 days and the expected yield is 0.5×10⁶-4×10⁶ neuronal lineage cellsper 1×10⁶ T-MSC after 4 weeks.

In another embodiment, T-MSC may be cultured to induce chondrogenicdifferentiation. T-MSC will be grown in a pellet in high glucose DMEMsupplemented with 0.5-10 mM (optimally 1 mM) Sodium Pyruvate, 0.05-1 mM(optimally 0.1 mM) ascorbic acid 2-phosphate, 0.05-1 μM (optimally 0.1μM) dexamethasone, 0.2-2% (optimally 1%) ITS, and 1-50 ng/ml (optimally10 ng/mL) TGF-β3. The medium is changed every 2 to 3 days and theexpected yield is 0.5×10⁶-4×10⁶ neuronal lineage cells per 1×10⁶ T-MSCafter 20 days.

In another embodiment, T-MSC may be cultured to induce myogenicdifferentiation. T-MSC will be grown in low-glucose DMEM supplementedwith 10% FBS, 1-20 μM (optimally 10 μM) 5-azacytidine, and 1-50 ng/ml(optimally 10 ng/ml) basic FGF. After 24 hours, the myogenic inductionmedium will be replaced with DMEM supplemented with 10% FBS plus 1-50ng/ml (optimally 10 ng/ml) basic FGF. The medium is changed every 2 to 3days and the expected yield is 0.5×10⁶-4×10⁶ neuronal lineage cells per1×10⁶ T-MSC after 2 weeks.

In another embodiment, T-MSC may be cultured to induce fibroblastdifferentiation. T-MSC will be grown in hMSCs that were treated withDMEM plus 10% FBS supplemented 50-200 ng/ml (optimally 100 ng/ml) ofrecombinant human Connective Tissue Growth Factor (CTGF) and 1-100 μg/ml(optimally 50 μg/ml) ascorbic acid. The medium is changed every a to 4days and the expected yield is 0.5×10⁶-4×10⁶ neuronal lineage cells per1×10⁶ T-MSC after 4 weeks.

All the cell lineages and cell types derived from T-MSC using anydifferentiation methods including, but not limited to, the methods aboveare called T-MSC-DL throughout.

Pharmaceutical Preparations

In one embodiment, provided herein is a pharmaceutical compositioncomprising a therapeutically effective amount of a T-MSC and apharmaceutically acceptable carrier.

The pharmaceutical compositions can comprise any number of T-MSC and/orT-MSC-DL. For example, a single unit dose of T-MSC can comprise, invarious embodiments, about, at least, or no more than 1×10⁵, 5×10⁵,1×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰,1×10¹¹ or more T-MSC and/or T-MSC-DL.

The pharmaceutical compositions disclosed herein comprise populations ofcells that comprise 50% viable cells or more (that is, at least 50% ofthe cells in the population are functional or living). Preferably, atleast 60% of the cells in the population are viable. More preferably, atleast 70%, 80%, 90%, 95%, or 99% of the cells in the population in thepharmaceutical composition are viable.

The pharmaceutical compositions disclosed herein can comprise one ormore compounds that, e.g., facilitate engraftment (e.g., anti-T-cellreceptor antibodies, an immunosuppressant, or the like); stabilizerssuch as albumin, dextran 40, gelatin, hydroxyethyl starch, and the like.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human, and approved by aregulatory agency of a Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. “Carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions in water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil, and the like. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents.

These compositions can take the form of solutions, suspensions,emulsions, tablets, pills, capsules, powders, sustained-releaseformulations, cachets, troches, lozenges, dispersions, suppositories,ointments, cataplasms (poultices), pastes, powders, dressings, creams,plasters, patches, aerosols, gels, liquid dosage forms suitable forparenteral administration to a patient, and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a patient.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable form ofcarrier so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

Pharmaceutical compositions adapted for oral administration may becapsules, tablets, powders, granules, solutions, syrups, suspensions (innon-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatincapsules may comprise lactose, starch or derivatives thereof, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, stearicacid or salts thereof. Soft gelatin capsules may comprise vegetableoils, waxes, fats, semi-solid, or liquid polyols. Solutions and syrupsmay comprise water, polyols, and sugars. An active agent intended fororal administration may be coated with or admixed with a material thatdelays disintegration and/or absorption of the active agent in thegastrointestinal tract. Thus, the sustained release may be achieved overmany hours and if necessary, the active agent can be protected fromdegradation within the stomach. Pharmaceutical compositions for oraladministration may be formulated to facilitate release of an activeagent at a particular gastrointestinal location due to specific pH orenzymatic conditions.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient over a prolonged period of time.

Pharmaceutical compositions adapted for nasal and pulmonaryadministration may comprise solid carriers such as powders which can beadministered by rapid inhalation through the nose. Compositions fornasal administration may comprise liquid carriers, such as sprays ordrops. Alternatively, inhalation directly through into the lungs may beaccomplished by inhalation deeply or installation through a mouthpiece.These compositions may comprise aqueous or oil solutions of the activeingredient. Compositions for inhalation may be supplied in speciallyadapted devices including, but not limited to, pressurized aerosols,nebulizers or insufflators, which can be constructed so as to providepredetermined dosages of the active ingredient.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injectable solutions orsuspensions, which may contain anti-oxidants, buffers, bacteriostats,and salutes that render the compositions substantially isotonic with theblood of the subject. Other components which may be present in suchcompositions include water, alcohols, polyols, glycerine, and vegetableoils. Compositions adapted for parental administration may be presentedin unit-dose or multi-dose containers, such as sealed ampules and vials,and may be stored in a freeze-dried (lyophilized) condition requiringonly the addition of a sterile carrier, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules, and tablets. Suitable vehicles that can beused to provide parenteral dosage forms of the invention are well knownto those skilled in the art. Examples include: Water for Injection USP;aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, and LactatedRinger's Injection; water-miscible vehicles such as ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Selection of a therapeutically effective dose will be determined by theskilled artisan considering several factors which will be known to oneof ordinary skill in the art. Such factors include the particular formof the inhibitor, and its pharmacokinetic parameters such asbioavailability, metabolism, and half-life, which will have beenestablished during the usual development procedures typically employedin obtaining regulatory approval for a pharmaceutical compound. Furtherfactors in considering the dose include the condition or disease to betreated or the benefit to be achieved in a normal individual, the bodymass of the patient, the route of administration, whether theadministration is acute or chronic, concomitant medications, and otherfactors well known to affect the efficacy of administered pharmaceuticalagents. Thus, the precise dose should be derided according to thejudgment of the person of skill in the art, and each patient'scircumstances, and according to standard clinical techniques.

In certain embodiments, patients are treated with antipyretic and/orantihistamine (acetaminophen and diphenhydramine hydrochloride) tominimize ally possible DMSO infusion toxicity related to thecryopreserve component in the hES-MSC treatment.

T-MSC Conditioned Media and Derivatives

The T-MSC disclosed herein can be used to produce conditioned mediumthat is immunosuppressive, that is, medium comprising one or morebiomolecules secreted or excreted by the stem cells that have adetectable immunosuppressive effect on a plurality of one or more typesof immune cells. In various embodiments, the conditioned mediumcomprises medium in which T-MSC have grown for at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or more days. In other embodiments, theconditioned medium comprises medium in which T-MSC have grown to atleast 30%, 40%, 50%, 60%, 70%, 80%, 90% confluence, or up to 100%confluence. Such conditioned medium can be used to support the cultureof a separate population of T-MSC, or stem cells of another kind. Inanother embodiment, the conditioned medium comprises medium in whichT-MSC have been differentiated into an adult cell type. In anotherembodiment, the conditioned medium of the invention comprises medium inwhich T-MSC and non-T-MSC have been cultured.

Thus, in one embodiment, the invention provides a composition comprisingculture medium, cell lysate and/or other derivatives from a culture ofT-MSC, wherein the T-MSC (a) adhere to a substrate (b) express CD73,CD105, CD90, CD29, CD44, CD145, IL-10, TGFb2, HGF, but do not expressIL-6, TNFα, IL-12 and/or RAGE. In another specific embodiment, thecomposition comprises an anti-proliferative agent, anti-MIP-1α oranti-MIP-1β antibody.

Provided herein is a method of using T-MSC as described herein as feedercells for bone marrow hematopoietic stem cell, peripheral bloodhematopoietic stem cell and umbilical-cord hematopoietic stem cellexpansion. In certain embodiments, the T-MSC suitable for the disclosedmethod express Stro-3, Stro-1, DL1, and/or Nestin. The T-MSC can also bemodified or engineered to express high level of Stro-3. Stro-1, DL1,Nestin or Frizzle using the method disclosed herein in Section 5.5. Incertain embodiments, T-MSC is co-cultured with bone marrow hematopoieticstem cells, peripheral blood hematopoietic stem cells and/orumbilical-cord hematopoietic stem cells. In certain embodiments, theT-MSC are mesenchymal stromal cells. Provided herein is a co-culture ofT-MSC as described herein and bone marrow hematopoietic stem cells.Provided herein is a co-culture of T-MSC as described herein andumbilical-cord hematopoietic stem cells.

Matrices Comprising T-MSC and/or T-MSC Derived Lineages

The invention further comprises matrices, hydrogels, scaffolds, and thelike that comprise T-MSC and/or T-MSC-DL, T-MSC and/or T-MSC-DL can beseeded onto a natural matrix, e.g., a biomaterial. In certainembodiments, the scaffold is obtained by 3D printing. The T-MSC and/orT-MSC-DL can be suspended in a hydrogel solution suitable for, e.g.,injection. Suitable hydrogels for such compositions includeself-assembling peptides, such as RAD16. In one embodiment, a hydrogelsolution comprising the cells can be allowed to harden, for instance ina mold, to form a matrix having cells dispersed therein forimplantation. T-MSC and/or T-MSC-DL in such a matrix can also becultured so that the cells are mitotically expanded prior toimplantation. The hydrogel is, e.g., an organic polymer (natural orsynthetic) that is cross-linked via covalent, ionic, or hydrogen bondsto create a three-dimensional open-lattice structure that entraps watermolecules to form a gel. Hydrogel-forming materials includepolysaccharides such as alginate and salts thereof, peptides,polyphosphazines, and polyacrylates, which are cross-linked ionically,or block polymers such as polyethylene oxide-polypropylene glycol blockcopolymers which are cross-linked by temperature or pH, respectively. Insome embodiments, the hydrogel or matrix of the invention isbiodegradable. In some embodiments of the invention, the formulationcomprises an in situ polymerizable gel (see, e.g., U.S. PatentApplication Publication 2002/0022676; Anseth et al., J. Control Release,78(1-3):199-209 (2002); Wang et al., Biomaterials, 24(22);3969-80(2003).

In some embodiments, the polymers are at least partially soluble inaqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. Examples of polymers having acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, polyvinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic add and vinyl ether monomers or polymers can alsobe used. Examples of acidic groups are carboxylic acid groups, sulfonicadd groups, halogenated (preferably fluorinated) alcohol groups,phenolic OH groups, and acidic OH groups.

The T-MSC, T-MSC-DL and/or co-cultures thereof can be seeded onto athree-dimensional framework or scaffold and implanted in viva. Such aframework can be implanted in combination with any one or more growthfactors, cells, drugs or other components that stimulate tissueformation or otherwise enhance or improve the practice of the invention.

Examples of scaffolds that can be used in the present invention includenonwoven mats, porous foams, or self-assembling peptides. Nonwoven matscan be formed using fibers comprised of a synthetic absorbable copolymerof glycolic and lactic acids (e.g., PGA/PLA) (VICRYL, Ethicon, Inc.,Somerville, N.J.). Foams, composed of, e.g.,poly(s-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed byprocesses such as freeze-drying, or lyophilization (see, e.g., U.S. Pat.No. 6,355,699), can also be used as scaffolds.

The T-MSC and/or T-MSC-DL can also be seeded onto, or contacted with, aphysiologically acceptable ceramic material including, but not limitedto, mono-, di-, tri-, alpha-tri-, beta-tri-, and tetra-calciumphosphate, hydroxyapatite, fluoroapatites, calcium sulfates, calciumfluorides, calcium oxides, calcium carbonates, magnesium calciumphosphates. biologically active glasses such as BIOGLASS®, and mixturesthereof. Porous biocompatible ceramic materials currently commerciallyavailable include SURGIBONE® (CanMedica Corp., Canada), ENDOBON® (MerckBiomaterial France, France), CEROS® (Mathys, AG, Bettlach, Switzerland),and mineralized collagen bone grafting products such as HEALOS™ (DePuy,Inc., Raynham, Mass.) and VITOSS®, RHAKOSS™, and CORTOSS® (Orthovita,Malvern, Pa.). The framework can be a mixture, blend or composite ofnatural and/or synthetic materials.

In another embodiment, T-MSC and/or T-MSC-DL can be seeded onto, orcontacted with, a felt, which can be, e.g., composed of a multifilamentyarn made from a bioabsorbable material such as PGA, PLA, PCL copolymersor blends, or hyaluronic acid.

The T-MSC and/or T-MSC-DL can, in another embodiment, be seeded ontofoam scaffolds that may be composite structures. Such foam scaffolds canbe molded into a useful shape, such as that of a portion of a specificstructure in the body to be repaired, replaced or augmented. In someembodiments, the framework is treated, e.g., with 0.1M acetic acidfollowed by incubation in polylysine, PBS, and/or collagen, prior toinoculation of the cells of the invention in order to enhance cellattachment. External surfaces of a matrix may be modified to improve theattachment or growth of cells and differentiation of tissue, such as byplasma-coating the matrix, or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, etc.), acellular matrix, and/or other materials such as, but not limited to,gelatin, alginates, agar, agarose, and plant gums, and the like.

In some embodiments, the scaffold comprises, or is treated with,materials that render it non-thrombogenic. These treatments andmaterials may also promote and sustain endothelial growth, migration,and extracellular matrix deposition. Examples of these materials andtreatments include but are not limited to natural materials such asbasement membrane proteins such as laminin and Type IV collagen,synthetic materials such as EPTFE, and segmented polyurethaneureasilicones, such as PURSPAN™ (The Polymer Technology Group, Inc.,Berkeley, Calif.). The scaffold can also comprise anti-thrombotic agentssuch as heparin; the scaffolds can also be treated to alter the surfacecharge (e.g., coating with plasma) prior to seeding with stem cells.

Immortalized T-MSC and/or T-MSC-DL

Mammalian T-MSC and/or T-MSC-DL can be conditionally immortalized bytransfection with any suitable vector containing a growth-promotinggene, that is, a gene encoding a protein that, under appropriateconditions, promotes growth of the transfected cell, such that theproduction and/or activity of the growth-promoting protein is relatableby an external factor. In a preferred embodiment the growth promotinggene is an oncogene such as, but not limited to, v-myc, N-myc, c-myc,p53, SV40 large T antigen, polyoma large T antigen, E1a adenovirus or E7protein of human papillomavirus.

External regulation of the growth-promoting protein can be achieved byplacing the growth-promoting gene under the control of anexternally-regulatable promoter, e.g., a promoter the activity of whichcan be controlled by, for example, modifying the temperature of thetransfected cells or the composition of the medium in contact with thecells. In one embodiment, a tetracycline (tet)-controlled geneexpression system can be employed (see Gossen et al., Proc. Natl. Acad.Sci. USA 89:5547-5551, 1992; Hoshimaru et al., Proc. Natl. Acad. Sci.USA 93:1518-1523, 1996). In the absence of tet, a tet-controlledtransactivator (tTA) within this vector strongly activates transcriptionfrom _(ph)CMV⁺-1, a minimal promoter from human cytomegalovirus fused totet operator sequences. tTA is a fusion protein of the repressor (tetR)of the transposon-10-derived tet resistance operon of Escherichia coliand the acidic domain of VP 16 of herpes simplex virus. Low, non-toxicconcentrations of tet (e.g., 0.01-1.0 μg/mL) almost completely abolishtransactivation by tTA.

In one embodiment, the vector further contains a gene encoding aselectable marker, e.g., a protein that confers drug resistance. Thebacterial neomycin resistance gene (neo) is one such marker that may beemployed within the present invention. Cells carrying neo^(R) may beselected by means known to those of ordinary skill in the art, such asthe addition of, e.g., 100-200 μg/mL G418 to the growth medium.

Transfection can be achieved by any of a variety of means known to thoseof ordinary skill in the art including, but not limited to, retroviralinfection. In general, a cell culture may be transfected by incubationwith a mixture of conditioned medium collected from the producer cellline for the vector and DMEM/F12 containing N2 supplements. For example,a stem cell culture prepared as described above may be infected after,e.g., five days in vitro by incubation for about 20 hours in one volumeof conditioned medium and two volumes of DMEM/F12 containing N2supplements. Transfected cells carrying a selectable marker may then beselected as described above.

Following transfection, cultures are passaged onto a surface thatpermits proliferation, e.g., allows at least 30% of the cells to doublein a 24 hour period. Preferably, the substrate is apolyornithine/laminin substrate, consisting of tissue culture plasticcoated with polyornithine (10 μg/mL) and/or laminin (10 μg/mL), apolylysine/laminin substrate or a surface treated with fibronectin.Cultures are then fed every 3-4 days with growth medium, which may ormay not be supplemented with one or more proliferation-enhancingfactors. Proliferation-enhancing factors may be added to the growthmedium when cultures are less than 50% confluent.

The conditionally-immortalized T-MSC and/or T-MSC-DL cell lines can bepassaged using standard techniques, such as by trypsinization, when80-95% confluent. Up to approximately the twentieth passage, it is, insome embodiments, beneficial to maintain selection (by, for example, theaddition of G418 for cells containing a neomycin resistance gene). Cellsmay also be frozen in liquid nitrogen for long-term storage.

Clonal cell lines can be isolated from a conditionally-immortalizedhuman T-MSC cell line prepared as described above. In general, suchclonal cell lines may be isolated using standard techniques, such as bylimiting dilution or using cloning rings, and expanded. Clonal celllines may generally be fed and passaged as described above.

Conditionally-immortalized human T-MSC cell lines, which may, but neednot, be clonal, may generally be induced to differentiate by suppressingthe production and/or activity of the growth-promoting protein underculture conditions that facilitate differentiation. For example, if thegene encoding the growth-promoting protein is under the control of anexternally-regulatable promoter, the conditions, e.g., temperature orcomposition of medium, may be modified to suppress transcription of thegrowth-promoting gene. For the tetracycline-controlled gene expressionsystem discussed above, differentiation can be achieved by the additionof tetracycline to suppress transcription of the growth-promoting gene.In general, 1 μg/mL tetracycline for 4-5 days is sufficient to initiatedifferentiation. To promote further differentiation, additional agentsmay be included in the growth medium.

Assays

The T-MSC and/or T-MSC-DL can be used in assays to determine theinfluence of culture conditions, environmental factors, molecules (e.g.,biomolecules, small inorganic molecules, etc.) and the like on stem cellproliferation, expansion, and/or differentiation, compared to T-MSCand/or T-MSC-DL not exposed to such conditions.

In a preferred embodiment, the T-MSC and/or T-MSC-DL are assayed forchanges in proliferation, expansion or differentiation upon contact witha molecule. In one embodiment, for example, the invention provides amethod of identifying a compound that modulates the proliferation of aplurality of T-MSC and/or T-MSC-DL, comprising contacting the pluralityof T-MSC and/or T-MSC-DL with the compound under conditions that allowproliferation, wherein if the compound causes a detectable change inproliferation of the T-MSC and/or T-MSC-DL compared to a plurality ofT-MSC and/or T-MSC-DL not contacted with the compound, the compound isidentified as a compound that modulates proliferation of T-MSC and/orT-MSC-DL. In a specific embodiment, the compound is identified as aninhibitor of proliferation. In another specific embodiment, the compoundis identified as an enhancer of proliferation.

In another embodiment, the invention provides a method of identifying acompound that modulates the expansion of a plurality of T-MSC and/orT-MSC-DL, comprising contacting the plurality of T-MSC and/or T-MSC-DLwith the compound under conditions that allow expansion, wherein if thecompound causes a detectable change in expansion of the plurality ofT-MSC and/or T-MSC-DL compared to a plurality of T-MSC and/or T-MSC-DLnot contacted with the compound, the compound is identified as acompound that modulates expansion of T-MSC and/or T-MSC-DL. In aspecific embodiment, the compound is identified as an inhibitor ofexpansion, in another specific embodiment, the compound is identified asan enhancer of expansion.

In another embodiment, disclosed herein is a method of identifying acompound that modulates the differentiation of a T-MSC and/or T-MSC-DL,comprising contacting a T-MSC and/or T-MSC-DL with a compound underconditions that allow differentiation, wherein if the compound causes adetectable change in differentiation of the T-MSC and/or T-MSC-DLcompared to a T-MSC and/or T-MSC-DL not contacted with the compound, thecompound is identified as a compound that modulates proliferation ofT-MSC and/or T-MSC-DL. In a specific embodiment, the compound isidentified as an inhibitor of differentiation. In another specificembodiment, the compound is identified as an enhancer ofdifferentiation.

Therapeutic Uses of Human Embryonic Stem Cell Derived Mesenchymal StemCells

Mesenchymal stem cells derived from bone marrow (BM-MSCs) have been usedas cell based therapy for T cell related autoimmune diseases, includingmultiple sclerosis (MS), but due to limited sources, unstable quality,and biosafety concerns of using cells derived from adult tissue, theiruse as a therapeutic aid has been limited.

The novel method for generating mesenchymal stem cells from embryonicstem cells set forth herein, and the novel T-MSC generated from thismethod, provide new therapies for T cell related autoimmune disease, inparticular multiple sclerosis.

In certain embodiments, T-MSC given to mice pre-onset of EAE, remarkablyattenuated the disease score of these animals. The decrease in score wasaccompanied by decreased demyelination, T cell infiltration, andmicroglial responses in the central nervous system, as well as repressedimmune cell proliferation, and differentiation in vitro.

In certain embodiments, a gradual decline of disease score in EAE miceafter treatment with T-MSC, post disease onset, was observed. In certainembodiments, T-MSC have both prophylactic and therapeutic effects on thedisease.

In certain embodiments, the immunosuppressive activity of the T-MSCaccount for the prophylactic effect on the disease as irradiated T-MSC,which are unlikely to replace damage myelin, and were also effective inreducing disease score. In one embodiment, irradiation does not shortenthe lifespan of the T-MSC.

In certain embodiments, the therapeutic effect of the T-MSC involveimmunosuppression as well as neural repair and regeneration.

In certain embodiment, EAE mice treated with T-MSC have much fewerinflammatory T cells in their central nervous system and less T cellsinfiltrating the spinal cord. The T-MSC can reduce damage and symptomscaused by inflammatory T cells, making them useful in therapy andprevention of all T cell related autoimmune diseases. T-MSC alsodecreased demyelination.

The characteristics of the T-MSC are all in marked contrast to theresults obtained with bone marrow-derived mesenchymal stem cells.BM-MSCs only suppressed mouse T cell proliferation in response toanti-CD3 stimuli at low doses in vitro, and even enhanced Th1 and Th17cell infiltration into the CNS. Autoreactive effector CD4⁺ T cells havebeen associated with the pathogenesis of several autoimmune disorders,including multiple sclerosis, Crohn's disease, and rheumatoid arthritis.These CD4⁺ T cells include Th1 and Th17 cells. There are only mild ornegligible effects of human BM-MSCs on EAE mice (Gordon et al. 2008a;Zhang et al. 2005; Payne et al. 2012). A recent report showed areduction of disease score of only 3.5 to 3.0 of ERE mice treated withhuman umbilical-derived MSCs (Liu et al. 2012). The results herein andthose from these studies highlight the novelty and usefulness of thedisclosed T-MSC.

Additionally, BM-MSC and T-MSC have very similar global transcriptionalprofiles, but differentially express some pro- and anti-inflammatoryfactors. Among them, IL-6 is expressed at a much higher level in BM-MSCsthan T-MSC. Moreover, IL-6 expression in BM-MSCs was double upon IFNγstimulation in vitro, whereas it remained low in the T-MSC.

IL-6 is a pleiotropic cytokine involved in crosstalk betweenhematopoietic/immune cells and stromal cells, including the onset andresolution of inflammation. IL-6 can promote the differentiation andfunctions of Th17 cells (Dong, 2008). The levels of IL-6 are elevated inmononuclear cells in blood and in brain tissue from MS patients(Patanella et al., 2010), as well as in serum in aged humans (Sethe etal., 2006). Mice lacking IL-6 receptor α at the time T cell priming areresistant to EAE (Leech et al., 2012). Site-specific production of IL-6in the CNS can re-target and enhance the inflammatory response in EAE(Quintana et al., 2009), whereas IL-6-neutralizing antibody can reducesymptoms in EAE mice (Gijbels et al., 1995). Thus, IL-6 has become apromising therapeutic target for treatment of MS.

Immunomodulation of peripheral T cell activity and regeneration andrepair of neural cells are widely recognized modes of MSC therapeuticaction in MS and in EAE (Al Jumah and Abumaree, 2012; Auletta et al.,2012; Morando et al., 2012). However, long-term functional neuronalrecovery and sustained disease remission in MS needs repair of thedamaged blood-brain barrier and blood-spinal cord barrier (Correale andVilla, 2007; Minagar et al., 2012). In other words, MS is aninflammatory, neurodegenerative, and vascular disease, and effectivetreatment need to target all three component.

The characteristics of T-MSC make them uniquely suited for the treatmentof T cell related autoimmune diseases especially multiple sclerosis. Inparticular, the T-MSC can decrease disease scores of EAE mice, but alsodecrease demyelination and decrease Th1 and Th17 proliferation, and havelow expression of IL-6. These latter two characteristics make themsuitable to treat other T cell related autoimmune diseases.Additionally, the ability of the T-MSC to cross the blood-brain barrierand blood-spinal cord barrier, makes them superior as a treatment andprevention of multiple sclerosis and other autoimmune diseases relatedto the central nervous system.

One embodiment provided herein is a method of treating or preventing a Tcell related autoimmune disease comprising the steps of administering atherapeutically effective amount of solution, cell culture orpharmaceutical preparation comprising T-MSC to the subject in needthereof. The T cell related autoimmune diseases would include but arenot limited to multiple sclerosis, inflammatory bowel disease, Crohn'sdisease, graft versus host disease, systemic lupus erythematosus, andrheumatoid arthritis. The subject is preferably a mammal, and mostpreferably human. The solation, cell culture or pharmaceuticalpreparation can comprise irradiated or non-irradiated T-MSC. Thesolution, cell culture or pharmaceutical preparation is preferablyadministered by injection.

Multiple sclerosis has been categorized into four subtypes:relapsing/remitting; secondary progressive; primary progressive; andprogressive relapsing. The relapsing/remitting subtype is characterizedby unpredictable relapses followed by long periods of remission.Secondary progressive MS often happens in individuals who start withrelapsing/remitting MS and then have a progressive decline with noperiods of remission. Primary progressive MS describes a small number ofindividuals who never have remission after their initial symptoms.Individuals with progressive relapsing, the least common subtype, have asteady neurologic decline, and suffer from acute attacks.

Provided herein is a method for treating or preventing multiplesclerosis disease in a subject in need thereof, comprising the steps ofadministering a therapeutically effective amount of solution, cellculture or pharmaceutical preparation comprising T-MSC as described inthe preceding paragraphs, to the subject in need thereof. The multiplesclerosis can be relapsing/remitting multiple sclerosis,progressive/relapsing multiple sclerosis, primary multiple sclerosis, orsecondary multiple sclerosis. The subject is preferably a mammal, andmost preferably human. The solution, cell culture or pharmaceuticalpreparation can comprise irradiated or non-irradiated T-MSC. Thesolution, cell culture or pharmaceutical preparation is preferablyadministered by injection.

Multiple sclerosis manifests in a variety of symptoms including sensorydisturbance of the limbs, optic nerve dysfunction, pyramidal tractdysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction,ataxia and diplopia attacks.

A further embodiment of the present invention is a method of treatingmultiple sclerosis comprising the steps of administering atherapeutically effective amount of solution, cell culture orpharmaceutical preparation comprising T-MSC, to the subject in needthereof, wherein there is detectable improvement in at least one ofthese symptoms, at least two of these symptoms. at least four of thesesymptoms, at least five of these symptoms or all of these symptoms.

The Expanded Disability Status Scale (EDSS) is the most commonly usedrating scale to evaluate the clinical status of patients with multiplesclerosis. It measures disability along several separate parameters:strength, sensation, brainstem functions (speech and swallowing),coordination, vision, cognition, and bowel/bladder continence. It is awell-accepted measure of disability in MS and it is not particularlydifficult or time consuming to perform. The EDSS quantifies disabilityin eight Functional Systems (FS) and allows neurologists to assign aFunctional System Score (FSS) in each of these (Kurtzke 1983).

Kurtzke defines functional systems as follows:

-   -   pyramidal    -   cerebellar    -   brainstem    -   sensory    -   bowel and bladder    -   visual    -   cerebral    -   other

The EDSS steps 1.0 to 4.5 refer to people with multiple sclerosis whoare fully ambulatory. EDSS steps 5.0 to 9.5 are defined by theimpairment to ambulation. The clinical meaning of each possible resultis the following:

-   -   0.0: Normal Neurological Exam    -   1.0: No disability, minimal signs on 1 FS.    -   1.5: No disability, minimal signs on 2 of 7 FS    -   2.0: Minimal disability in 1 of 7 FS    -   2.5: Minimal disability in 2 FS    -   3.0: Moderate disability in 1 FS; or mild disability in 3-4 FS,        though fully ambulatory    -   3.5: Fully ambulatory but with moderate disability in 1 FS and        mild disability in 1 or 2 FS; or moderate disability in 2 FS; or        mild disability in 5 FS    -   4.0: Fully ambulatory without aid, up and about 12 hrs a day        despite relatively severe disability. Able to walk without aid        500 meters    -   4.5: Fully ambulatory without aid, up and about much of day,        able to work a full day, may otherwise have some limitations of        full activity or require minimal assistance. Relatively severe        disability. Able to walk without aid 300 meters    -   5.0: Ambulatory without aid for about 200 meters. Disability        impairs full daily activities    -   5.5: Ambulatory for 100 meters, disability precludes full daily        activities    -   6.0: Intermittent or unilateral constant assistance (cane,        crutch or brace) required to walk 100 meters with or without        resting    -   6.5: Constant bilateral support (cane, crutch or braces)        required to walk 20 meters without resting    -   7.0: Unable to walk beyond 5 meters even with aid, essentially        restricted to wheelchair, wheels self, transfers alone; active        in wheelchair about 12 hours a day    -   7.5: Unable to take more than a few steps, restricted to        wheelchair, may need aid to transfer; wheels self, but may        require motorized chair for full day's activities    -   8.0: Essentially restricted to bed, chair, or wheelchair, but        may be out of bed much of day; retains self-care functions,        generally effective use of arms    -   8.5: Essentially restricted to bed much of day, some effective        use of arms, retains some self-care functions    -   9.0: Helpless bed patient, can communicate and eat    -   9.5: Unable to communicate effectively or eat/swallow    -   10.0: Death due to MS

Provided herein is a method for treating multiple sclerosis disease in asubject in need thereof, comprising the steps of administering atherapeutically effective amount of solution, cell culture orpharmaceutical preparation comprising T-MSC, to the subject in needthereof wherein the subject demonstrates improvement on the ExpandedDisability Status Scale of at least one point, and preferably at leasttwo points.

There are other therapeutic agents that have been used to treat andprevent multiple sclerosis, including but not limited to, fingolimod,adrenocorticotropic hormone (ACTH), methylprednisolone, dexamethasone,IFNβ-1a, IFN-1b, gliatriamer acetate, cyclophosphamide, methotrexate,azathioprine, cladribine, cyclosporine, mitoxantrone, and sulfasalazine.

Therefore, the method of the present invention can further comprise the:administration of one or more additional therapeutic agents to thesubject, including but not limited to, fingolimod, adrenocorticotropichormone (ACTH), methylprednisolone, dexamethasone, IFNβ-1a, IFN-1b,gliatriamer acetate, cyclophosphamide, methotrexate, azathioprine,cladribine, cyclosporine, mitoxantrone and sulfasalazine. In a furtherembodiment, these additional therapeutic agents can be administeredprior to, after, or at the same time as the T-MSC, or can be conjugatedor attached to the T-MSC.

Other than T cells, T-MSC also have strong suppressive function on Bcells, dendritic cells, neutrophils, NK cells, macrophage and otherinflammatory and immunity related functions, Thus, T cell, B cell,inflammatory and/or innate immunity related autoimmune diseases that canall be treated by the disclosed T-MSC include, hut are not limited to,Alopecia Areata, Anklosing Spondylitis, Antiphospholipid Syndrome,Autoimmune Addison's Disease, Autoimmune Hemolytic Anemia, AutoimmuneHepatitis, Autoimmune Inner Ear Disease, Autoimmune LymphoproliferativeSyndrome (ALPS), Autoimmune Thrombocytopenic Purpura (ATP), Behcet'sDisease, Bullous Pemphigoid, Cardiomyopathy, Celiac Sprue-Dermatitis,Chronic Fatigue Syndrome Immune Deficiency Syndrome (CFIDS), ChronicInflammatory Demyelinating Polyneuropathy, Chronic Obstructive PulmonaryDisease (COPD), Cicatricial Pemphigoid, Cold Agglutinin Disease, CRESTSyndrome, Crohn's Disease, Dego's Disease, Dermatomyositis,Dermatomyositis—Juvenile, Discoid Lupus, Essential MixedCryoglobulinemia, Fibromyalgia—Fibromyositis, Grave's Disease,Guillain-Barre, Hashimoto's Thyroiditis, idiopathic Pulmonary Fibrosis,idiopathic Thrombocytopenia Purpura (ITP), IgA Nephropathy, InsulinDependent Diabetes (Type I), Type II diabetes, Juvenile Arthritis,Lupus, Meniere's Disease, Mixed connective Tissue Disease, MultipleSclerosis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Anemia,Polyarteritis Nodosa, Polychondritis, Polyglancular Syndromes,Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, PrimaryAgammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud'sPhenomenon, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis,Sarcoidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syndrome,Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, UlcerativeColitis, Uveitis, Vasculitis, Vitiligo, Wegener's Granulomatosis, or anyacute or chronic inflammation related to burning, surgery, injury, andallergy.

T-MSC can be differentiated into multiple cell lineages including, butnot limited to, adipocytes, myoblast cells, neural lineage cells,osteoblast cells, fibroblasts, chondrocytes, and stromal cells. Thesecells derived from T-MSC (T-MSC-DL) can be used to treat multiple tissueinjury, and can be used for tissue engineering, tissue repair, tissueregeneration purposes like, joint healing, tendon healing, connectivetissue healing, neural lineage tissue and cells healing, fat tissuehealing, bone healing, skin healing, other wound healing, musclehealing, cartilage healing, smooth muscle healing, myocardiac healing,epithelia tissue healing, ligament healing, stroma repair, etc.

Specifically, T-MSC can be differentiated into neural lineage cells,which can be used to treat many neural disease including but not limitedto Agraphia, Alzheimer's disease, Amyotrophic lateral sclerosis,Aphasia, Apraxia, Arachnoiditis, Ataxia Telangiectasia, Attentiondeficit hyperactivity disorder, Auditory processing disorder, Autism,Alcoholism, Asperger's syndrome, Bipolar disorder, Bell's palsy,Brachial plexus injury, Brain damage, Brain injury, Brain tumor, Canavandisease, Capgras, Causalgia, Central pain syndrome, Central pontinemyelinolysis, Centronuclear myopathy, Cephalic disorder, Cerebralaneurysm, Cerebral arteriosclerosis, Cerebral atrophy, Cerebralgigantism, Cerebral palsy, Cerebral vasculitis, Cervical spinalstenosis, Charcot-Marie-Tooth disease, Chiari malformation, Chorea,Chronic fatigue syndrome, Chronic inflammatory demyelinatingpolyneuropathy (CIDP), Chronic pain, Coffin-Lowry syndrome, Coma,Complex regional pain syndrome, Compression neuropathy, Congenitalfacial diplegia, orticobasal degeneration, Cranial arteritis,Craniosynostosis, Creutzfeldt-Jakob disease, Cumulative traumadisorders, Cushing's syndrome, Cytomegalic inclusion body disease(CIBD), Cytomegalovirus Infection, Dandy-Walker syndrome, Dawsondisease, De Morsier's syndrome, Dejerine-Klumpke palsy, Dejerine-Sottasdisease, Delayed sleep phase syndrome, Dementia, Dermatomyositis,Developmental dyspraxia, Diabetic neuropathy, Diffuse sclerosis, Downssyndrome, Dravet syndrome, Dysautonomia, Dyscalculia, Dysgraphia,Dyslexia, Dystonia, Empty sella syndrome, Encephalitis, Encephalocele,Encephalotrigeminal angiomatosis, Encopresis, Epilepsy, Erb's palsy,Erythromelalgia, Essential tremor, Fabry's disease, Fahr's syndrome,Fainting, Familial spastic paralysis, Febrile seizures, Fisher syndrome,Friedreich's ataxia, Fibromyalgia, Foville's syndrome, Fetal AlcoholEffect Gaucher's disease, Gerstmann's syndrome, Giant cell arteritis,Giant cell inclusion disease, Globoid Cell Leukodystrophy, Gray matterheterotopia, Guillain-Barré syndrome, HTLV-1 associated myelopathy,Hallervorden-Spatz disease, Head injury, Headache, Hemifacial Spasm,Hereditary Spastic Paraplegia, Heredopathia atactica polyneuritiformis,Herpes zoster oticus, Herpes zoster, Hirayama syndrome,Holoprosencephaly, Huntington's disease, Hydranencephaly, Hydrocephalus,Hypercortisolism, Hypoxia, Immune-Mediated encephalomyelitis, Inclusionbody myositis, Incontinentia pigmenti, Infantile phytanic acid storagedisease, Infantile Refsum disease, Infantile spasms, Inflammatorymyopathy, intracranial cyst, Intracranial hypertension, Joubertsyndrome, Karak syndrome, Kearns-Sayre syndrome, Kennedy disease,Kinsbourne syndrome, Klippel Feil syndrome, Krabbe disease,Kugelberg-Welander disease, Lafora disease, Lambert-Eaton myasthenicsyndrome, Landau-Kleffner syndrome, Lateral medullary (Wallenberg)syndrome, Learning disabilities, Leigh's disease, Lennox-Gastautsyndrome, Lesch-Nyhan syndrome, Leukodystrophy, Lewy body dementia,Lissencephaly, Locked-In syndrome, Lou Gehrig's disease (See amyotrophiclateral sclerosis), Lumbar disc disease, Lumbar spinal stenosis, Lymedisease—Neurological Sequelae, Machado-Joseph disease (Spinocerebellarataxia type 3), Macrencephaly, Macropsia, Megalencephaly,Melkersson-Rosenthal syndrome, Menieres disease, Meningitis, Menkesdisease, Metachromatic leukodystrophy, Microcephaly, Micropsia,Migraine, Miller Fisher syndrome, Mini-stroke (transient ischemicattack), Misophonia, Mitochondrial myopathy, Mobius syndrome, Monomelicamyotrophy, Motor Neurone Disease—see amyotrophic lateral sclerosis,Motor skills disorder Moyamoya disease, Mucopolysaccharidoses,Multi-infarct dementia, Multifocal motor neuropathy, Multiple sclerosis,Multiple system atrophy, Muscular dystrophy, Myalgic encephalomyelitis,Myasthenia gravis, Myelinoclastic diffuse sclerosis, MyoclonicEncephalopathy of infants, Myoclonus, Myopathy, Myotubular myopathy,Myotonia congenita, Narcolepsy, Neurofibromatosis, Neuroleptic malignantsyndrome, Neurological manifestations of AIDS, Neurological sequelae oflupus, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migrationdisorders, Neurosis, Niemann-Pick disease, Non 24-hour sleep-wakesyndrome, Nonverbal learning disorder, Naurological disorder,O'Sullivan-McLeod syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara syndrome, Olivopontocerebellar atrophy,Opsoclonus myoclonus syndrome, Optic neuritis, Orthostatic Hypotension,Otosclerosis, Overuse syndrome, Palinopsia, Paresthesia, Parkinson'sdisease, Paramyotonia Congenita, Paraneoplastic diseases, Paroxysmalattacks, Parry-Romberg syndrome, Pelizaeus-Merzbacher disease, PeriodicParalyses, Peripheral neuropathy, Pervasive developmental disorders,Photic sneeze reflex, Phytanic acid storage disease, Pick's disease,Pinched nerve, Pituitary tumors, PMG, Polyneuropathy, Polio,Polymicrogyria, Polymyositis, Porencephaly, Post-Polio syndrome,Postherpetic Neuralgia (PHN), Postural Hypotension, Prader-Willisyndrome, Primary Lateral Sclerosis, Prion diseases, Progressivehemifacial atrophy, Progressive multifocal leukoencephalopathy,Progressive Supranuclear Palsy, Pseudotumor cerebri, Quadriplegia,Rabies, Ramsay Hunt syndrome type I, Ramsay Hunt syndrome type II,Ramsay Hunt syndrome type III—see Ramsay-Hunt syndrome, Rasmussen'sencephalitis, Reflex neurovascular dystrophy, Refsum disease, Repetitivestress injury, Restless legs syndrome, Retrovirus associated myelopathy,Rett syndrome, Reye's syndrome, Rhythmic Movement Disorder, Rombergsyndrome, Saint Vitus dance, Sandhoff disease, Schilder'sdisease[disambiguation needed], Schizencephaly, Sensory integrationdysfunction, Septo-optic dysplasia, Shaken baby syndrome, Shingles,Shy-Drager syndrome, Sjögren's syndrome, Sleep apnea, Sleeping sickness,Snatiation, Solos syndrome, Spasticity, Spina bifida, Spinal cordinjury, Spinal cord tumors, Spinal muscular atrophy, Spinocerebellarataxia, Split-brain, Steele-Richardson-Olszewski syndrome, Stiff-personsyndrome, Stroke, Sturge-Weber syndrome, Subacute sclerosingpanencephalitis, Subcortical arteriosclerotic encephalopathy,Superficial siderosis, Sydenham's chorea, Syncope, Synesthesia,Syringomyelia, Tarsal tunnel syndrome, Tardive dyskinesia, Tardivedysphrenia, Tarlov cyst, Tay-Sachs disease, Temporal arteritis, Tetanus,Tethered spinal cord syndrome, Thomsen disease, Thoracic outletsyndrome, Tic Douloureux, Todd's paralysis, Tourette syndrome, Toxicencephalopathy, Transient ischemic attack, Transmissible spongiformencephalopathies, Transverse myelitis, Traumatic brain injury, Tremor,Trigeminal neuralgia, Tropical spastic paraparesis, Trypanosomiasis,Tuberous sclerosis, Ubisiosis, Template:Unipolar depression, VonHippel-Lindau disease (VHL), Viliuisk Encephalomyelitis (VE),Wallenberg's syndrome, Werdnig-Hoffman disease, West syndrome, Whiplash,Williams syndrome, Wilson's disease.

Uses of T-MSC as Delivery Systems

Because it has been shown that the T-MSC of the present invention havethe unique ability to cross the blood-brain barrier and the blood-spinalcord barrier, a further embodiment of the present invention is a methodof using T-MSC for delivery of agents through the blood brain barrierand/or the blood spinal cord barrier, by attaching or conjugating theagent to the T-MSC to form a complex; and administering the T-MSC-agentcomplex to a subject, wherein the T-MSC cross the blood-brain and/or theblood-spinal cord barrier and deliver the agent to the central nervoussystem. The T-MSC may be in the form of a single cell, a cell culture, asolution or a pharmaceutical preparation. Agents would include but arenot limited to chemicals, drugs, proteins, DNA, RNA, antibodies, andsmall molecules.

A further embodiment of the present invention is a delivery system forthe delivery of agents through the blood brain barrier and/or the bloodspinal cord barrier comprising T-MSC and an agent conjugated or attachedto the T-MSC.

The ability to permeate the blood-brain barrier and the blood-spinalcord barrier would be useful in the treatment and prevention of diseasesincluding but not limited to neurological disorders, multiple sclerosis,cancer, Parkinson's Disease, Alzheimer's Disease, Huntington's Disease,meningitis, encephalitis, rabies, epilepsy, dementia, Lyme's Disease,stroke, and amyotrophic lateral sclerosis, as well as brain and spinalcord injury. Thus, a subject in need thereof would have a disease or beat risk for a disease in which the blood-brain barrier and/orblood-spinal cord barrier is involved. Thus, a further embodiment of thepresent invention is a method of treating a disease or injury, byattaching or conjugating an agent to the T-MSC to form a complex; andadministering the T-MSC-agent complex to a subject in need thereof,wherein the T-MSC cross the blood-brain and/or the blood-spinal cordbarrier and deliver the agent to the central nervous system, and theagent is used as a treatment or prevention of the disease or injury ofthe subject. Since the T-MSC have strong migration ability andinfiltration ability, it can also been used as carrier for tumor/cancertherapy to carry anti-tumor drugs and proteins. The T-MSC may be in theform of a single cell, a cell culture, a solution or a pharmaceuticalpreparation. Agents include, but are not limited to, chemicals, drugs,proteins, DNA, RNA, micro-RNA, non-coding RNA, antibodies, smallmolecules and/or nano particles.

Agents that are useful in the treatment and prevention of diseasesinclude, but ARE not limited to, antibiotics, anti-viral agents,anti-fungal agents, steroids, chemotherapeutics, anti-inflammatories,cytokines, and/or synthetic peptides.

Proteins and peptides would also be useful to conjugate to the T-MSC andwould include erythropoietin (EPO), anti-beta-amyloid peptides, tissueplasminogen activator (TPA), granulocyte colony stimulating factor(G-CSF), interferon (IFN), growth factor/hormone, anti-VEGF peptides,anti-TNF peptides, NGF, HGF, IL-2, CX3CL1, GCV, CPT-11, cytosinedeaminase, HSV-TK, carboxyesterase, oncolytic virus, TSP-1, TRAIL, FASL,IL-10, and TGFb. Proteins and peptides that bind to particular receptorsand block these receptors would also be useful and are contemplated bythe current invention to be attached to the T-MSCs.

DNA and RNA that coded for therapeutic proteins and peptide would alsobe useful to conjugate to the T-MSC for delivery across the blood-brainbarrier and/or the blood-spinal cord barrier.

The terms “antibody” and “antibodies” include polyclonal antibodies,monoclonal antibodies, humanized or chimeric antibodies, single chain Fvantibody fragments, Fab fragments, and F(ab′)₂, fragments. Polyclonalantibodies are heterogeneous populations of antibody molecules that arespecific for a particular antigen, while monoclonal antibodies arehomogeneous populations of antibodies to a particular epitope containedwithin an antigen. Monoclonal antibodies are particularly useful in thepresent invention.

Any agent that would block the activation, expression and/or action of amolecule or the receptor of the molecule in the pathway related to anydisease in which crossing the blood-brain barrier and/or blood-spinalcord barrier is useful could be attached or conjugated to the T-MSCs.Such agents include but are not limited to chemicals, phytochemicals,pharmaceuticals, biologics, small organic molecules, antibodies, nucleicacids, peptides, and proteins.

Inhibiting a pathway can also be effected using “decoy” molecules whichmimic the region of a target molecule in the pathway binds andactivates. The activating molecule would bind to the decoy instead ofthe target, and activation could not occur.

Inhibition can also be effected by the use of a “dominantly interfering”molecule, or one in which the binding portion of activating molecule isretained but the molecule is truncated so that the activating domain islacking. These molecules would bind to receptors in the pathway but beunproductive and block the receptors from binding to the activatingmolecule. Such decoy molecules and dominantly interfering molecule canbe manufactured by methods known in the art, and attached or conjugatedto the T-MSC for delivery across the blood-brain or blood-spinal cordbarrier.

A method for delivery of agents across the blood-brain and/orblood-spinal cord barrier is also useful for diagnostic agents,including but not limited to chemicals, antibodies, peptides, proteins,DNA, and RNA. Such agents in order to be useful for diagnosis must havea means of being visualized and/or quantified. Such means include, butare not limited to, fluorescence, biomarkers, dyes, radioactive isotypeslabels and/or nanoparticles.

Such a method for delivery and a delivery system would be useful for thediagnosis of neurological disorders, multiple sclerosis, cancer,Parkinson's Disease, Alzheimer's Disease, Huntington's Disease,meningitis, encephalitis, rabies, epilepsy, dementia, Lyme's Disease,stroke, and amyotrophic lateral sclerosis, as well as brain and spinalcord injury. Thus, a further embodiment of the present invention is amethod of diagnosing a disease or injury, by attaching or conjugatingthe agent to the T-MSC to form a complex; and administering theT-MSC-agent complex to a subject in which a disease is suspected,wherein the T-MSC cross the blood-brain and/or the blood-spinal cordbarrier and deliver the agent to the central nervous system. The T-MSCmay be in the form of a single cell, a cell culture, a solution or apharmaceutical preparation. Agents would include but are not limited tochemicals, drugs, proteins, DNA, RNA, antibodies, and small molecules.

Agents, no matter the type and whether for treatment, prevention, ordiagnosis, can be conjugated or attached to the T-MSC by any methodknown in the art including, but not limited to, synthetic extracellularmatrix, alginate-poly-L-Lysine encapsulate and/or container.

In certain embodiments, large scale production at industrial level ofmanufacturing is included in the present disclosure, methods of whichare well known in the art. In certain embodiments, the large scaleproduction includes the use of a Hyper-STACK 2D culture system and/or aMicrocarrier 3D bioreactor.

EXAMPLES Example 1 Derivation of T-MSC Material and Methods

The following reagents and materials were obtained from thebelow-described sources:

-   -   Customized mTeSR1 Medium: Stem Cell Technology, Inc.    -   BMP4: Stemgent or other vendors    -   SB431542: Cayman Chemical or other vendors    -   A83-01: Stemgent or other vendors.    -   ALK5 inhibitor: Stemgent or other Vendors    -   DMEM/F12: GIBCO Life Technologies    -   alpha-MEM: GIBCO Life Technologies    -   Fetal Bovine Serum: GIBCO Life Technologies or other vendors

CT2 hESC line derived at the University of Connecticut Stem Cell Corewas cultured for two passages on irradiated mouse embryonic fibroblast(MEF) as feeders. The hESCs were then split on plates coated withMatrigel (BD Biosciences, San Jose, Calif.) and cultured in mTeSR1(Ludwig et al., 2006) (Stem Cell Technologies, Vancouver, Canada),ESI-017, ESI-051, ESI-053, ESI-049, and ESI-35 human embryonic stemcells were purchased from BioTime, Inc. (CA).

Derivation of T-MSC

As shown in FIG. 1, hESCs at ˜80% confluency on the Matrigel-coatedplates were digested with Dispase at 1 mg/ml for 5-10 min. The cellswere then washed with mTESR1 medium once and split as small clumps orsingle cells onto Matrigel-coated plate and cultured in mTeSR1 for 12hr. Then the culture medium was replaced by a trophoblast-formationmedium containing BMP4 (2-100 ng/ml), or optional A83-01 (0.1-1 μM).After culture for 48-72 hr, the cells changed from hESC-like morphologyinto trophoblast-like morphology featured by flat, enlarged deli size,small nuclear/cytosol ratio, and diffuse cell borders. The cells weredigested with Tryp-LE and washed with MSC growth medium (alpha-MEMcontaining 20% fetal bovine serum and non-essential amino acids). Thecells were then plated onto Matrigel-coated plates at a density of 5,000cells/cm². The medium was changed after 24 hr, and then changed every3-4 days. After 6 more days, the cells were differentiated intospindle-like cells similar to the morphology of typical MSCs. Morphologyof Day2 Trophablast are shown in FIG. 2A, morphology of Day 5 pre-T-MSCare shown in FIG. 2B, morphology of T-MSC are shown in FIG. 2C.

Derivation of HB-MSC

CT2 hESC cells were differentiated into EB cells and then enriched forHB as previously described (Lu et al., 2008); Lu et al., 2007)) 50-80%confluent hEC cell on the Matrigel plate were digested with Dispase (1mg/ml for 5 to 10 minutes) and then washed with EB formation basalmedium, HPGM (Lonza, Walksville, Md.), or STEMLINE I/II HematopoieticStell Cell Expansion Medium (Sigma, St. Louis, Mo.), or StemSpan H3000(Stem Cell Technologies, Vancouver, Canada), or IMDM with 10% FBS, orDMEDM/F12 with 10% FBS. Cells were then cultured in EB formation mediumsupplemented with 50 ng/ml of VEGF (Peprotech) and 50 ng/ml of BMP4(Stemgent) for 48 hours on ultra-low plate at a density of about 2-3million cells/ml. After 48 hours, half the culture medium was replacedwith fresh EB formation medium plus 25-50 ng/ml of bFGF.

Four days later, EB cells formed in the medium were harvested anddissociated into single cells with TrypLE (Invitrogen) at 37° C. for 2-3minutes. Cells were washed and resuspended at 1-5 million cells/ml in ESformation basal medium. The single cell suspension was then mixed at1:10 with Hemangioblast Growth Medium (Stem Cell Technologies,Vancouver, Canada).

Blast cell growth medium (BGM) were made as follows: To 100 mlSerum-free methylcellulose CFU medium (Stem Cell Technologies, H4436 orH4536). added with VEGF, TPO and FLT3-Ligand to 50 ng/ml, bFGF to20-50ng/ml, 1 ml of EX-CYTE Growth Enhancement Media Supplement and 1 mlof Pen/Strap, mix well.

The mixtures were vortexed and plated onto ultra ow plates by passingthrough a 16G needle and cultured for 5-9 days at 37° C. with 5% CO₂.

Single cells were then resuspended in MSC medium containing: 1) 10-20%FBS in alpha-MEM (Invitrogen) or 2) 10-20% KOSR alpha-MEM, 3) 10-20% FBSDMEM high-glucose, or 4) 10-20% KOSR DMEM high-glucose, and cultured oneither Matrigel, gelatin, vitronectin, laminin, fibronectin, or collagenI coated plates at a density of 100-5,000 cell/cm². The medium waschanged after 24 hours and refreshed every 2-4 days. After 6-12 days thecells gradually differentiated into spindle-like cells similar totypical MSCs.

Derivation of MSC through SB431542

This method was published previously (Chen et al., 2012).

Results

It was found that the method generated T-MSC that have superiorefficiency, yield and purity. As shown in the bottom panel of FIG. 1, onDay 10, T-MSC already generated >90% purify of MSC with 10 fold cellnumber increase, whereas other methods either did not have any MSC oronly had very low purity of MSCs. On Day20 T-MSC already had 3000 fadexpansion with >99% purity of MSCs, whereas the other methods onlyexpanded 20 fold at most. By day 30, 0.1 million of hESC generated 50billion of T-MSC, that is a 500,000 fold expansion of the originalhESCs, whereas the other methods only expanded 3000 fold at most.

Example 2 Characterization of T-MSC Cells

The T-MSC cells obtained in Example 1 were further analyzed using flowcytometry immunofluorescence staining.

Materials and Methods

Flow cytometry staining was used to characterize the T-MSCs. Cells werewashed and blocked with 2% BSA in PBS, and stained with antibodies forvarious cell surface markers Trop-2 (Trp-2, eBioscience), CD31, CD34,CD29, CD73, CD90, CD105, CD44, CD45, CD146, CD166, HLA-ABC, HLA-DR,HLA-G (BD Bioscience or eBioscience) by following the manufacturers'instructions. Data were collected on FAGS LSR II Flow Cytometer usingFACSDiva software (BD Bioscience). Post-acquisition analysis wasperformed with the FlowJo software (Treestar).

Results

The attached cells obtained from Day 2 trophoblast. Day 5 pre-T-MSC andDay 9 T-MSC were stained with CD73 and Trop-2. The trophoblast cellsonly expressed high levels of Trop-2 (greater than 95%3, but less than1% of CD73 (FIG. 3A); the pre-T-MSC at day 5 has more than 50% of cellsexpress both Trop-2 and CD73, 40% of the cells express only CD73 (FIG.3B); T-MSC at day 9 of hESC differentiation has less than 1% of thecells express Trop-2, and 99% of cells express only CD73 (FIG. 3C).

Further characterization of the T-MSC by FACS staining of multiple cellsurface markers show T-MSC express <3% of Trop-2, <1% of CD31,CD34. >99% of CD73, >95% of CD90, >90% of CD105, >99% of CD44 and >80%of CD29 (FIGS. 4A-H).

Example 3 T-MSCs Have a Stronger Inhibition on T Cell Functions in Vitrothan BM-MSC

hEs-MSCs and BM-MSCs were compared for their ability to inhibit T cellproliferation in vitro following antigen stimulation.

Materials and Methods Culture of BM-MSCs

BM-MSCs were derived from BM mononuclear cells (BMMNCs) or obtained fromAllCells, Inc. (Alameda) and Lonza (Basel, Switzerland) BMMNCs. Forderivation, BMMNCs were thawed and plated onto tissue culture plasticdishes in MEM+20% FBS. Adherent cells began to appear within the first4-5 days and fed every 3 days until day ˜10-12, when cells wereharvested and replated at 3,000-5,000 cells/cm².

The in vitro assay for T cell proliferation was performed usinglymphocytes isolated from mouse peripheral lymph nodes. Theselymphocytes were labeled with 5 μM of carboxyfluorescein succinimidylester (CFSE) to track their proliferation by monitoring CFSE dilution intheir daughter cells, for 10 minutes at 37° C. 10,000 T-MSCs or BM-MSCswere mixed with 100,000 lymphocytes per well in a 96-well plate, and thecells were stimulated for proliferation with plate-bound anti-CD3 (at0.3, 1 μg/ml) and soluble anti-CD28 antibodies (1 μg/ml, eBioscience,CA). The cells were collected 3 days after the stimulation, followed byFACS staining with anti-CD4 and anti-CD8 antibodies (BD Bioscience, CA).CFSE dilution was gated on CD4+ and CD8+ T cells, respectively.

Results

Using the in vitro assay with mouse lymphocytes, it was found T-MSCsinhibited the proliferation of mouse CD4+ and CD8+ T cells whenstimulated with anti-CD3 antibody at 0.3 and 1 μg/ml, whereas BM-MSConly did so when the T cells were stimulated with anti-CD3 antibody atlow doses, i.e., 0.3 μg/ml (FIG. 5)

Example 4 T-MSCs Attenuate the Disease Score of EAE Mice

Because it has been shown that BM-MSCs can attenuate the diseaseprogression of the mouse model of multiple sclerosis, experimentalautoimmune encephalomyelitis (EAE), the T-MSCs obtained in Example 1were injected into mice with EAE to determine if they would have thesame effect.

Materials and Methods

Derivation of MSC through SB431542: the MSC derived from this methodwill be called hES-MSC(SB). This Method was published previously (Chenet al., 2012).

The mouse EAE model was induced as previously described (Stromnes andGoverman, 2006). C57BL/6 mice were subcutaneously injected with amixture of myelin oligodendrocyte glycoprotein peptide 35-55 (MOG³⁵⁻⁵⁵),Freund's adjuvant, and pertussis toxin contained in the EAE inductionKit (Hooke Laboratories, Inc, MA. (Cat. # EK-0114)) following themanufacturer's protocol and as described in Ge et al. (2012).

BM-MSC, T-MSC or hES-MSC(SB) at 10⁶ cells/mouse or PBS (a vehiclecontrol) was intraperitoneal (i.p.) injected on day 6 (for pre-onset) or18 (for post-onset) after the immunization. The disease score wasmonitored on the mice every day for up to 31 days.

The disease scoring system is as follows:

-   -   0: no sign of disease;    -   1: loss of tone in the tail;    -   2: partial hind limb paralysis;    -   3: complete hind limb paralysis;    -   4: front limb paralysis; and    -   5: moribund

(Stromnes and Goverman, 2006),

Results

As shown in FIG. 6, the T-MSCs significantly attenuated the dailydisease scores when injected at 6 days or pre-onset of disease, showinga prophylactic effect of the T-MSCs. Mice injected with BM-MSC did notattenuate the disease score, hES-MSC(SB) had a partial effect inattenuating the disease score but not as good as T-MSC.

Example 5 Multi-Lineage Differentiation of T-MSC Materials and MethodsOsteogenesis, Chondrogenesis and Adipogenesis of T-MSC

STEMPRO Osteogenesis and Chondrogenesis Differentiation Kits(Invitrogen, Grand Island, N.Y.) were used for osteogenesis andchondrogenesis, and the Hyclone AdvanceSTEM Adipogenic Differentiationkit (Thermo Scientific, Logan, Utah) for adipogenesis, following themanufacturers' instructions.

Results

As shown in FIG. 7, T-MSC had good potency in differentiating into allthe 3 lineages of mesoderm tissues, osteoblasts, chondrocyte andadipocytes. Thus, T-MSC can be used as source for tissue regeneration,tissue engineering and tissue repair.

Example 6 T-MSC are Different from hES-HB-MSC and BM-MSC

Microarray analysis was performed to compare the gene expression profileof T-MSC, hES-HB-MSC and BM-MSCs.

Materials and Methods

For microarray analysis, RNA of hES-MSC at passages 2-4 or BM-MSC atpassage 3 were harvested with Trizol (Invitrogen, CA) following themanufacturer's protocol. The HumanHT-12 v4 Expression BeadChip(Illumina, San Diego, Calif.) was used to analyze the gene expressionprofile of the cells. Data were analyzed using Genome Studio V2011.1.Two BM-MSC cell lines from different sources were used, and two hES-MSCcell lines, derived from H9 and MA09, were used.

Results

As shown in FIG. 8, the overall expressional profiles of some keycytokines, transcription factors, cell surface markers are verydifferent between these 3 different MSCs. T-MSC may play different rolesin immunosuppression and tissue regeneration.

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While the disclosure has been described with reference to preferredembodiments, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted for theelements thereof without departing from the scope of the disclosure. Inaddition, many modifications may be made to adapt the teaching toparticular use, application, manufacturing conditions, use conditions,composition, medium, size, and/or materials without departing from theessential scope and spirit of the disclosure. Therefore, it is intendedthat the disclosure not be limited to the particular embodiments andbest mode contemplated for carrying out as described herein. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references cited herein are incorporated by reference in theirentireties and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

1-133. (canceled)
 134. A population of trophoblast-derived MSCs(T-MSCs), adipocytes, chondrocytes, osteoblast cells, neural lineagecells, myoblast cells, stromal cells and fibroblast cells produced bythe method comprising: a. culturing human embryonic stem cells in aserum free medium comprising at least one GSK3 inhibitor at aconcentration ranging from 0.05 μM to 0.2 μM, wherein the humanembryonic stem cells are cultured in the absence of feeder cells; b.culturing the cells from step a) in a serum-free medium comprisingvascular endothelial growth factor (VEGF) and bone morphogenic protein 4(BMP4) in an amount sufficient to induce formation of embryoid bodiescomprising human hemangio-colony forming cells; c. adding at least onegrowth factor to the culture resulting from step b), wherein the growthfactor is in an amount sufficient to expand human hemangio-colonyforming cells; d. disaggregating the hemangio-colony forming cellsresulting from step c) into single cells; and e. culturing the singlehemangio-colony forming cells resulting from step d) in mesenchymal stemcell medium containing serum, knockout serum replacement (KOSR), or in aserum-free medium to induce differentiation of the single cells intohuman mesenchymal stem cells; wherein at least 90% of the hES-MSCsexpress CD73, and said hES-MSCs comprise greater than 95% of cellsexpressing CD73, CD90, CD105, CD146, CD166, and CD44; (ii) comprisegreater than 80% of cells expressing CD13, CD29, CD54, and CD49E; (iii)comprise less than 5% of cells expressing CD45, CD34, CD31 and SSEA4;(iv) express IL-10 and TGFβ; (v) comprise less than 2% of cellsexpressing IL-6, IL-12 and TNFα; and (vi) comprise less than 0.001% ofcells co-expressing OCT4, NANOG, TRA-1-60 and SSEA4.
 135. Apharmaceutical composition comprising the population of T-MSCs of claim134 and a pharmaceutically acceptable carrier.
 141. A method fortreating a T cell, B cell, inflammatory and/or innate immunity relateddiseases comprising administering to a subject in need thereof thepopulation of T-MSCs of claim 134 in an amount sufficient to ameliorateor alleviate at least one symptom of the diseases, reverse the diseases,minimize the extent of the disease or hinder its development.
 142. A kitcomprising the population of T-MSCs of claim 134 and a carrier.
 143. Thekit of claim 142 further comprising a thawing reagent, immunosuppressiveenhancer, and anti-histamine.
 144. A method of delivering an agentthrough the blood-brain barrier and/or the blood-spinal cord barrier,said method comprising the steps of: attaching the agent to a hES-MSCfrom the population of hES-MSC of claim 134 to form a hES-MSC-agentcomplex; and administering the hES-MSC-agent complex to a subject inneed thereof, wherein the hES-MSC is capable of crossing the blood-brainbarrier and/or the blood-spinal cord barrier and the agent is for thetreatment, prevention or diagnosis of a disease or injury in the subjectin need thereof.
 145. A method for preventing or treating a T celland/or B cell related autoimmune disease comprising administering to asubject in need thereof the population of hES-MSC of claim 134, anamount sufficient to ameliorate or alleviate at least one symptom of theT cell related autoimmune disease or reverse the T cell and /or B cellrelated autoimmune disease.
 146. A method for preventing or treating amultiple sclerosis comprising administering to a subject in need thereofthe population of hES-MSC of claim 134, an amount sufficient toameliorate or alleviate at least one symptom of the disease or reversethe disease.
 147. A method for tissue regeneration or repair comprisingadministering to a subject in need thereof the population of T-MSCs ofclaim 134 in an amount sufficient to promote tissue regenerationsincluding, but not limited to, joint regeneration, tendon regeneration,connective tissue regeneration, neural lineage cells regeneration, fattissue regeneration, bone regeneration, skin regeneration, muscleregeneration, cartilage regeneration, smooth muscle regeneration,cardiac muscle regeneration, epithelia tissue regeneration, and/orligament regeneration.